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DNA nanotechnology as a tool to manipulate lipid bilayer membranesMeng, Zhuojun

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DNA nanotechnology as a tool to

manipulate lipid bilayer membranes

Zhuojun Meng

DNA nanotechnology as a tool to manipulate lipid bilayer membranes

Zhuojun Meng

PhD thesis

University of Groningen

October 2017

Zernike Institute PhD thesis series 2017-22

ISSN: 1570-1530

ISBN: 978-90-367-9976-8 (printed version)

ISBN: 978-90-367-9975-1 (electronic version)

The research described in thesis was carried out in Polymer Chemistry and

Bioengineering group at Zernike Institute for Advanced Materials,

University of Groningen, The Netherlands. This work was financially

supported by the Chinese Scholarship Council (CSC), the University of

Groningen and the Netherlands Organization for Science Research (NWO).

Cover design by: Zhuojun Meng

Printed by: Ridderprint BV

DNA nanotechnology as a tool to manipulate lipid bilayer membranes

PhD thesis

to obtain the degree of PhD at the University of Groningen on the authority of the

Rector Magnificus Prof. E. Sterken and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on

Friday 13 October 2017 at 16.15 hours

by

Zhuojun Meng

born on 5 May 1987 in Henan, China

Supervisor

Prof. A. Herrmann

Assessment committee

Prof. S. Vogel

Prof. A. M. van Oijen

Prof. D. J. Slotboom

Dedicated this book to myself

and my best friend

Qing Liu

Contents

Chapter 1

Functionalization of Lipid Bilayer Membranes ......................................................... 9

1. 1 Lipid bilayer membranes ................................................................................... 10

1.2 Classification and Preparation of Liposomes .............................................. 12

1.3 Modification and Applications of liposomes ............................................... 14

1.4 Motivation and Thesis Overview ...................................................................... 24

References ......................................................................................................................... 26

Chapter 2

Stability Study of Lipid-DNA on the Liposomal Membrane ............................... 31

2.1 Introduction .............................................................................................................. 32

2.2 Results and Discussion ......................................................................................... 35

2.3 Conclusion ................................................................................................................. 42

2.4 Experimental Section ............................................................................................ 42

References ......................................................................................................................... 48

Chapter 3

Efficient Fusion of Liposomes by Nucleobase Quadruple-Anchored DNA .. 51

3.1 Introduction .............................................................................................................. 52


3.3 Conclusion ................................................................................................................. 63


References ......................................................................................................................... 69

Chapter 4

DNA Replacement and Hybridization Chain Reaction on the Surface of

Liposome Membrane ......................................................................................................... 73

4.1 Introduction .............................................................................................................. 74



References ......................................................................................................................... 88

Chapter 5

Performing DNA Nanotechnology Operations on a Zebrafish Surface ......... 91

5.1 Introduction .............................................................................................................. 92


5.3 Conclusion ................................................................................................................ 101

5.4 Experiment Section .............................................................................................. 103

References ....................................................................................................................... 105

Summary ............................................................................................................................. 108

Samenvatting .................................................................................................................... 114

Acknowledgements ....................................................................................................... 119

Chapter 1

Functionalization of

Lipid Bilayer Membranes

Chapter 1

10

1. 1 Lipid bilayer membranes

Lipids play an important role in the physiology and pathophysiology of

living systems why they are produced, transported, and recognized by the

concerted actions of numerous enzymes, binding proteins, and receptors.1

Micelles are formed by the aggregation of single-chain lipids in a polar

solvent (such as water) beyond a particular concentration, known as

Critical Micelle Concentration (CMC) (Fig. 1.1A). Therefore, the micelle

formation and stability are highly dependent on the lipid concentration and

solvent composition (Fig. 1.1B).

Two-chain lipids can hardly be packed into micelles due to the bulky

hydrophobic part. They usually form a lipid bilayer membrane, which is a

thin polar sheet made of two layers of lipid molecules and is characterized

by hydrophobic tails facing inwards towards each other and hydrophilic

head groups facing outwards to associate with aqueous solution.2 At this

moment, the hydrophobic parts of the molecules are still in contact with

water, which leads to an energetically unfavorable state of the bilayer. This

is overcome through folding of the bilayer membrane into a liposome with

closed edges (Fig. 1.1C).3,4

Functionalization of Lipid Bilayer Membranes

11

Fig. 1.1 (A) Surface tension as a function of the surfactant concentration. Schematic structure

of a micelle (B) and a liposome (C). (Fig. 1.1 C was adapted from reference 4)

Chapter 1

12

1.2 Classification and Preparation of Liposomes

Depending on the number of bilayers, liposomes can be classified into two

categories: unilamellar vesicles (ULV) and multilamellar vesicles (MLV).

Unilamellar vesicles can also be classified into three categories on the basis

of their sizes, which can vary from nanometer to micrometer range: small

unilamellar vesicles (SUV), large unilamellar vesicles (LUV) and giant

unilamellar vesicles (GUV). GUVs also include other morphologies such as

multilamellar vesicles (MLV), which consist of SUVs or multiple concentric

bilayers (Fig. 1.2).

Fig. 1.2 Schematic structure of unilamellar and multilamellar liposomes.

There are four classical methods to prepare liposomes, differing in the way

how the lipids are dried from organic solvent and then redispersed in

aqueous buffer.5 These steps can be performed individually or jointly.6

These four methods are:

1. Hydration of a Thin Lipid Film.7

2. Reverse-Phase Evaporation Technique. 8

3. Solvent (Ether or Ethanol) Injection Technique.9,10

4. Detergent Dialysis.11


13

Since the “Hydration of a Thin Lipid Film” method, is widespread used and

easy to handle, it is explained here in more details. Firstly, the lipids are

dissolved and mixed in an organic solvent to assure a homogeneous

mixture. Once the lipids are thoroughly dispersed in the organic solvent,

the solvent is removed using a dry nitrogen stream in a fume hood to yield

a lipid film. The lipid film is dried to remove residual organic solvent by

using a vacuum desiccator overnight. Afterwards, hydration of the dry lipid

film is accomplished by stirring in an aqueous buffer. The temperature of

the hydrating buffer should be higher than the gel-liquid crystal transition

temperature (Tc) of the lipid. Subsequently, several stirring (above the Tc)

and freeze-thawing cycles of the swelling multilayer sample results in

MLVs. Finally, the sample is extruded multiple times using an extruder and

polycarbonate membranes to obtain unilamellar vesicles (LUVs or SUVs).

Fig. 1.3 shows the classical hydration method of liposome preparation.

Fig. 1.3 Schematic diagram of liposome preparation method. (Schematic obtained from www.

avantilipids.com)

Chapter 1

14

1.3 Modification and Applications of liposomes

The first discovery of liposomes in 1964 by A. D. Bangham12 was the

starting point for these self-assembled containers to become a

multifunctional tool in biology, biochemistry and medicine today. Because

of the structure, charge, chemical composition and colloidal size can be well

controlled by preparation methods, liposomes can be useful in various

applications. Vesicles can also be prepared from natural substances and are

therefore in many cases nontoxic, biodegradable, biocompatible, targetable

and non-immunogenic.13 Due to these properties, liposomes can be used as

drug14-16 protein, plasmid17 and gene18-21 delivery vehicles in medicine and

diagnosis.

1.3.1 Loading and surface modification

Molecular interactions between the cargo and the lipid bilayer membrane

play an important role on liposome formation and cargo encapsulation.22

Liposomes consist of an aqueous core surrounded by a lipid bilayer,

sectioning off two separate inner areas. They can carry hydrophobic

molecules in their hydrocarbon tail region (between the phospholipid

bilayer), or hydrophilic molecules in the core and direct the cargo to the

required diseased site in the body with some targeting moieties on the

surface.23 The thickness of the lipid bilayer is around 4 to 10 nm, which is a

natural barrier for many substances such as sugars and proteins.24 But

small hydrophilic substances such as water, gases, ammonia and glycerol

can penetrate freely through the bilayer.25-27 Some large hydrophilic

substances can be encapsulated in the water core of the liposome during

liposome preparation using the common thin layer hydration method.

Cationic liposomes, which are made of positively charged lipids, appear to

be better suited for DNA delivery due to the natural charge-charge

interaction between the positively charged lipid head groups and the

negatively charged phosphate groups of the DNA-backbone.28,29 Due to

their favorable interactions with negatively charged DNA and cell

membranes,30-33 cationic liposome–DNA complexes are increasingly being

researched for their use in gene therapy and nucleic acid release.34,35 In

order to increase liposomal drug accumulation in the desired cells and


15

tissues, the use of targeted liposomes with surface modification has been

suggested.

Surface modification of liposomes with controlled propertied requires the

chemical conjugation of peptides, DNA, antibodies or other targeting

molecules. Moreover, some “smart” vesicle designs allow the release of the

encapsulated cargo by incorporation of transport channels.36-39 Both

chemical attachment and physical interactions can be used to achieve

surface modification (Fig. 1.4A).

Fig. 1.4 (A) Schematic representation of liposomes surface modifications. (B) Interaction of the

particle with cell surface antigens and receptors.40 (C) Scheme of tetrac tagged liposome and

enhanced delivery by the ligand-mediated targeting strategy.41 (Fig. 1.4 B was adapted from

reference 40. Fig. 1.4 C was adapted from reference 41)

To realize active targeting, the liposome surface can be coated with ligands

or antibodies that will confer cell type-specificity to ensure that the

liposomes are internalized and that their content is released, improving the

efficacy and reducing side effects over non-targeted cells (Fig. 1.4B).40 For

instance, tetraiodothyroacetic acid (tetrac), a small molecule which binds

Chapter 1

16

to integrin αvβ3, was used for the surface modification of liposomes and

successfully enhanced the tumor-targeting ability of PEGylated liposome

(Fig. 1.4C).41 Although the physical properties of liposomes were not

significantly changed, tetrac-tagged liposomes showed significantly higher

cancer cell localization than the unmodified PEGylated liposome, and

tumor growth was effectively retarded. The ligand-mediated targeting

strategy could provide better therapeutic effects with more accurate

delivery of nanoparticles.

1.3.1.1 Membrane fusion

Surface modification of lipid bilayers can also be used for membrane fusion

which is an essential process of life resulting in the highly regulated

transport of bio-molecules both between and within cells.42-44 Membrane

fusion is an essential but not a spontaneous process as free energy is

required to overcome the electrostatic and steric repulsions between two

merging membrane surfaces and to break the hydration shell.45,46 A highly

conserved protein machinery, known as SNARE proteins (soluble N-

ethylmaleimide sensitive factor attachment protein receptors), facilitates

the communication within a cell.47-49 The SNAREs from synaptic vesicles

interact with the SNAREs from the target membrane to form a coiled-coil

bundle of four helices, pulling the membranes tightly together and

initiating fusion.

Design and construction of simplified artificial model systems mimicking

natural systems are one of the most promising approaches for studying

complex biological mechanisms.50 Several of these systems have been

reported for realizing membrane fusion, such as DNA51-53, peptides54, 55,

enzymes56 and polymers57. Yang et al. designed an artificial biorthogonal

targeting system that was able to target liposomes and other nanoparticles

efficiently to the tissue of interest by using coiled coil forming peptides,

E4[(EIAALEK)4] (E4) and K4[(KIAALKE)4] (K4) (Fig. 1.5C), which are

known to trigger liposomal membrane fusion when tethered to lipid

vesicles in the form of lipopeptides.58 The same group proved that E4

peptide-modified liposomes could deliver far-red fluorescent dye TOPRO-3

iodide (E4-Lipo-TP3) and doxorubicin (E4-Lipo-DOX) into HeLa cells


17

expressing K4 peptide (HeLa-K) on the surface. Then, E4-Lipo-TP3 and E4-

Lipo-DOX were injected into zebrafish xenografts of HeLa-K (Fig. 1.5A, B).

The results showed that E4-liposomes delivered TP3 to the implanted

HeLa-K cells (Fig. 1.5D), and E4-Lipo-DOX could suppress cancer

proliferation in the xenograft when compared to nontargeted conditions.

These data demonstrated that coiled-coil formation enables drug

selectivity and efficacy in vivo.

Fig. 1.5 Drug Delivery by E4/K4 Coiled-Coil Formation in Cells (A) and Zebrafish (B). (C)

Schematic representation of coiled-coil structure between peptides E and K. (D) E4/K4 coiled-

coil formation allows delivering the content in the liposome to cancer cells in the xenograft

zebrafish.58 (This figure was reproduced with permission from reference 58)

1.3.1.2 Controlled release

Conventional liposomes (Fig. 1.6A) are easily recognized by the

mononuclear phagocyte system and are rapidly cleared from the blood

stream.59 Many methods have been suggested to achieve long circulation of

liposomes in vivo by modification of the liposomal surface with hydrophilic

Chapter 1

18

polymers to delay the elimination process, such as coating the surface of

liposomes with biocompatible polymers like poly(ethylene glycol) (PEG)

linked phospholipids. These can be incorporated into the liposomal bilayer

to form a hydrophilic polymer shield over the liposome surface, protecting

the liposome from penetration or disintegration by plasma proteins60-65

(Fig. 1.6B). While many varieties have been synthesized by using

chemically modified forms of PEG, in some cases it’s necessary to make the

liposomes shed their cloak of modified PEG molecules when they reach

their target (Fig. 1.6C). In this way they can interact with the target and

release their payload. Using imaging technologies, visual evidence of the

effect of PEGylation on the circulation kinetics of the liposomes was

provided (Fig. 1.6D).66 The images clearly demonstrate that PEGylation

significantly enhances the persistence of liposomes in the blood stream. At

the same time, the uptake of PEGylated liposomes in organs (liver and

spleen) responsible for particle clearance decreased.

Fig. 1.6 Schematic representation of (A) conventional liposome, (B) PEG-liposome and (C)

chemically modified PEG-liposome. (D) The effect of PEGylation on the circulation persistence

of liposomes. The liposomes were labeled with Tc-99m, administered in rats, and the rats were

imaged with a gamma camera over 24 h. As is evident from the heart (H) image signal, the

PEG-liposomes remained in circulation even 24 h post-injection. The accumulation in the liver

(L) and the spleen (S) was also lower in the case of PEG-liposomes, as compared to the plain

liposomes.66 (Fig. 1.6 D was adapted from reference 66)


19

Ligands conjugated with hydrophobic molecules form amphiphiles. The

hydrophobic part can insert into the liposome bilayer, exposing the ligand

outside of the liposomes for being recognized or for other interactions. For

instance, DNA-b-polypropyleneoxide (DNA-ppo) has proven to be stably

anchored into the lipid membrane for over at least 24 h. In this way, the

containers are encoded with sequence information. The DNA-ppo present

on the surface was used for anchoring a photosensitizer by hybridization.

Upon light irradiation the PPO was oxidized leading to cargo release (Fig.

1.7).67

Fig. 1.7 Illustration of selective cargo release from DNA block copolymer (DBC) -decorated

phospholipid vesicles. (1) DNA-ppo is stably anchored in unilamellar lipid vesicles; (2) DBC-

decorated vesicles are functionalized with conjugated DNA-photosensitizers by hybridization;

(3) singlet oxygen is generated by light irradiation; and (4) selective cargo release is induced

by the oxidative effect of singlet oxygen.67 (This figure was reproduced with permission from

reference 67)

1.3.2 Stimuli-responsive liposomes

Liposomes can suspend cargos with their peculiar solubility properties and

act as a sustained-release system for microencapsulated molecules. After

modification, liposomes can be used as stimuli-responsive nanoparticles,

which are visionary concepts to deliver and release a drug exactly where it

is needed.68,69 There are several ways to trigger cargo release, such as

light,70 temperature71,72 and magnetism.73 Often two or more triggers need

Chapter 1

20

to be combined to appropriately improve the cargo release kinetics and

distribution to reduce side effects.

1.3.2.1 Light responsive vesicle systems

Methods of sensitizing liposomes to light have progressed from the use of

organic molecule moieties to the use of metallic plasmon resonant

structures which can be broadly categorized as photochemical or

photophysical release. Photochemical release can be achieved via

photoisomerization, photocleavage and photopolymerization, which all

lead to destabilization of the liposome bilayer and release of encapsulated

contents (Fig. 1.8A-C).

Fig. 1.8 Release from liposomes mediated by photochemical responses: photoisomerization (A),

photocleavage (B), or photopolymerization (C); and photophysical responses: molecular

absorbers (D) and gold nanoparticles (E).


21

On the other hand, photophysical release from liposomes does not rely on

any chemical changes of structures within or associated with the bilayer

membrane. Examples of photophysical release discussed here take

advantage of photothermal conversion of absorbed light with ensuing

thermal and/or mechanical processes in the lipid membrane and the

surrounding medium. The methods for achieving photophysical release are

developed around molecular absorbers (Fig. 1.8D) or gold nanoparticles

(Fig. 1.8E).70

1.3.2.2 Temperature responsive vesicle systems

Temperature-responsive liposomes are classified into two types:

traditional temperature-responsive liposomes and liposomes modified

with temperature-responsive polymers. Traditional temperature-

responsive liposomes which are composed of temperature-responsive

lipids show the greatest permeation of the lipid membrane at its gel-to-

liquid crystalline phase transition temperature.

Moreover, liposomes modified with temperature-responsive polymers

exhibit a lower critical solution temperature (LCST) behavior. These

polymers are soluble in an aqueous solution below this temperature but

dehydrate and aggregate if heated above the LCST. This behavior induces

the release of a drug within a polymer-modified liposome. For instance, a

temperature-responsive polymer, poly (N-isopropylacrylamide)-co-N,N'-

dimethylaminopropylacrylamide (P(NIPAAm-co-DMAPAAm)) was

synthesized and used for liposome modification. This research showed that

the polymer underwent dehydration and aggregation above 40 °C and that

temperature-responsive polymer-modified liposomes had faster cellular

uptake and release compared to non-modified liposomes (Fig. 1.9).74

Chapter 1

22

Fig. 1.9 Liposomes modified with temperature-responsive polymers are used for cellular

uptake. The copolymer displayed a thermosensitive transition at a lower critical solution

temperature (LCST) that is higher than body temperature. Above the LCST, the temperature-

responsive liposomes started to aggregate and release their content. The liposomes showed a

fixed aqueous layer thickness (FALT) at the surface below the LCST, and the FALT decreased

with increasing temperature. Above 37°C, cytosolic release from the temperature-responsive

liposomes was higher than that from the PEGylated liposomes, indicating intracellular

uptake.74 (This figure was adapted from reference 74)

1.3.2.3 Magnetic responsive vesicle systems

Magnetoliposomes are composed of a lipid bilayer surrounding

superparamagnetic iron oxide nanoparticles. Due to the biocompatibility,

size, material-dependent physicochemical properties and potential

applications as alternative contrast enhancing agents for magnetic

resonance imaging, magnetoliposomes are ideal candidates to achieve a

spatial and temporal control over drug release.75,76 Superparamagnetic iron

oxide nanoparticles (SPION) can be guided to their site of action using an

externally applied magnetic field. The subsequent accumulation of SPION

in the target site can be exploited for simultaneous drug delivery, MR

imaging or hyperthermia therapy of cancer (Fig. 1.10).


23

Fig. 1.10 Superparamagnetic iron oxide nanoparticles can be guided to the site of action using

an externally applied magnetic field.77 (This figure was adapted from reference 77)

In the beginning, liposomes were studied only for their physicochemical

properties as models of membrane morphology. Today, they are used as

delivery devices to encapsulate cosmetics, drugs, fluorescent detection

reagents, and as vehicles to transport nucleic acids, peptides, and proteins

to specific cellular sites in vivo. Advances in therapeutic applications of

liposomes have been achieved through surface modifications. With these

surface modifications, their biological stability could be increased, which

includes reduced constituent exchange and leakage as well as reduced

unwanted uptake by cells of the mononuclear phagocytic system.78

Targeting components such as antibodies can be attached to liposomal

surfaces and were used to create large antigen-specific complexes. In this

sense, liposomal derivatives are being used to target cancer cells in vivo, to

enhance detectability in immunoassay systems.

Chapter 1

24

1.4 Motivation and Thesis Overview

The overall goal of the work described in this thesis was to use DNA

nanotechnology as a tool to manipulate lipid bilayer surfaces. Our group

synthesized and characterized a new family of DNA amphiphiles containing

modified nucleobases. The modification is introduced in uracil and consists

of hydrophobic moieties. Through solid phase synthesis, the modified

nucleotides can be incorporated in any desired position and several

modifications per DNA strands can be introduced.79 The resulting DNA

sequences still undergo specific Watson-Crick base pairing. This property

combined with the amphiphilic nature of this lipid-DNA qualifies the

material as appealing candidate to interact with and manipulate biological

membrane structures.

In chapter 2, a powerful new approach was introduced by modifying DNA

with lipid chains at four nucleobases to tightly anchor the nucleotide to the

lipid membrane. This strategy allows highly stable incorporation of DNA

into the liposomal bilayer, thereby limiting dissociation. Several assays

were employed proving the incorporation and stable anchoring in the

phospholipid bilayer. These measurements involve small vesicles and

fluorescence energy transfer. These experiments allow to measure how

long the DNA amphiphiles remain in the bilayer.

In chapter 3, efficient fusion of liposomes was studied using lipid-DNA

introduced in the chapter before. While the orientation of DNA

hybridization played a significant role in the efficacy of full fusion of DNA-

grafted vesicles, the number of anchoring units was found to be a crucial

factor as well. As compared to vesicles functionalized with single-anchored

or double-anchored DNA, liposomes containing quadruple-anchored

oligonucleotides were found to be highly fusogenic, achieving considerable

full fusion of up to 29% without notable leakage. This study demonstrates

the importance of the DNA-anchoring strategy in hybridization-induced

vesicle fusion, as not only the structural properties of the unit itself, but

also the number of anchoring units determines its favorable fusion-

inducing properties. Several fluorescence assays, dynamic light scattering

and cryogenic transmission electron microscopy were utilized to prove

these results.


25

In chapter 4, we expand the functionality of DNA encoded vesicles

significantly. It was demonstrated that strand replacement can be carried

out. In this chapter it will be outlined what sequences and what DNA

amphiphiles are needed to reach this goal, i.e. changing the surface

functionalities of liposomes by the simple addition of oligonucleotides.

Moreover, it will be detailed how such a surface modification can be

amplified by a simple DNA-triggered supramolecular polymerization.

In chapter 5, we investigated whether it is possible to insert the lipid-

modified DNA sequences into the membrane of live zebrafish to function as

artificial receptor. We demonstrate that oligonucleotides functionalized

with a membrane anchor can be immobilized on a zebrafish. Protruding

single-stranded DNA atop the fish was functionalized by Watson-Crick base

pairing employing complementary DNA sequences. In this way, small

molecules and liposomes were guided and attached to the fish surface. The

anchoring process can be designed to be reversible allowing exchange of

surface functionalities by simple addition of DNA sequences. To achieve

this on a fish surface, the strand exchange experiments established in

chapter 4 on simple vesicles as model were crucial. Finally, a DNA based

amplification process was performed atop of the zebrafish enabling the

multiplication of surface functionalities from a single DNA anchoring unit.

Chapter 1

26

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28. Chonn, A.; Cullis, P. R.; Devine, D. V.; THE ROLE OF SURFACE CHARGE IN THE ACTIVATION AND ALTERNATIVE PATHWAYS OF COMPLEMENT BY LIPOSOME. J Immunol 1991, 46, 4234-4241.

29. Eastman, S. J.; Siegel, C.; Tousignant, J.; Smith, A.E.; Cheng, S.H.; Scheule, R.K.; Biophysical characterization of cationic lipid: DNA complexes. Biochim. Biophys. Acta 1997, 1325, 41-62.

30. Leventis, R.; Silvius, J. R.; Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles. Biochim. Biophys. Acta 1990, 1023, 124-132.

31. Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielson, M.; Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413-7417.

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33. Wrobel, I.; Collins, D.; Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta 1995, 1235, 296-304.

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38. Birkner, J. P.; Poolman, B.; Koҫer, A.; Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 12944-12949.

39. Louhivuori, M.; Risselada, H. J.; Giessen, van der E.; Marrink, S. J.; Release of content through mechano-sensitive gates in pressurized liposomes. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 19856-19860.

40. Yhee, J. Y.; Lee, S.; Kim, K.; Advances in targeting strategies for nanoparticles in cancer imaging and therapy. Nanoscale 2014, 6, 13383.

41. Lee, S.; Kim, J.; Shim, G.; Kim, S.; Han, S. E.; Kim, K.; Kwon, I. C.; Choi, Y.; Kim, Y. B.; Kim, C.; Oh, Y.; Tetraiodothyroacetic acid-tagged liposomes for enhanced delivery of anticancer drug to tumor tissue via integrin receptor. J Control Release 2012, 164, 213-220.

42. Ma, M.; Bong, D.; Controlled Fusion of Synthetic Lipid Membrane Vesicles. Acc Chem Res. 2013, 46, 2988-2997.

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44. Kong, L.; Askes, S. H. C.; Bonnet, S.; Kros, A.; Campbell, F.; Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes. Angew. Chem. Int. Ed. 2016, 55, 1396-1400.

45. Chernomordik, L. V.; Kozlov, M. M.; Protein-lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 2003, 72, 175-207.

46. Cohen, F.S.; Melikyan, G. B.; The energetics of membrane fusion from binding, through hemifusion, pore formation and pore enlargement. J. Membr. Biol. 2004, 199, 1-14.

47. Weber, T.; Zemelman, B. V.; Mcnew, J. A.; Westermann, B.; Gmachl, M.; Parlati, F.; Söllner, T. H.; Rothman, J. E.; SNAREpins: minimal machinery for membrane fusion. Cell 1998, 92, 759-772.

48. Jahn, R.; Scheller, R. H.; SNAREs–engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 2006, 7, 631-643.

49. Hong, W. J.; Lev, S.; Tethering the assembly of SNARE complexes. Trends Cell Biol. 2014, 24, 35-43.



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51. Stengel, G.; Zahn, R.; Höök, F.; DNA-induced programmablefusionof phospholipidvesicles. J. Am. Chem. Soc. 2007, 129, 9584-9585.

52. Chan, Y. H. M.; van Lengerich, B.; Boxer, S. G.; Lipid-anchored DNAmediates vesicle fusion as observed by lipid content mixing. Biointerphases 2008, 3,17-21.

53. Chan, Y. H. M.; van Lengerich, B.; Boxer, S. G.; Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotide. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 979-984.

54. Zheng, T.; Voskuhl, J.; Versluis, F.; Zope, H. R.; Tomatsu, I.; Marsden, H. R.; Kros, A. Controlling The Rate of Coiled Coil Driven Membrane Fusion. Chem. Commun. 2013, 49, 3649-3651.

55. Kong, L.; Askes, S. H.; Bonnet, S.; Kros, A.; Campbell, F. Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes. Angew. Chem. Int. Ed. 2016, 55, 1396-1400.

56. Mukai, M.; Sasaki, Y.; Kikuchi, J.; Fusion-Triggered Switching of Enzymatic Activity on an Artificial Cell Membrane. Sensors 2012, 12, 5966-5977.

57. Su, W.; Luo, Y.; Yan, Q.; Wu, S.; Han, K.; Zhang, Q.; Gu, Y.; Li, Y.; Photoinduced Fusion of Micro-Vesicles Self-Assembled from Azobenzene-Containing Amphiphilic Diblock Copolymers. Macromol. Rapid Commun. 2007, 28, 1251-1256.

58. Jian Yang, Yasuhito Shimada, René C. L. Olsthoorn, B. Ewa Snaar-Jagalska, Herman P. Spaink, and Alexander Kros, Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model. ACS Nano. 2016, 10, 7428-7435.

59. Nag, O. K.; Awasthi, V.; Surface Engineering of Liposomes for Stealth Behavior. Pharmaceutics 2013, 5, 542-569.

60. Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Sterically stabilized liposomes-improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11460-11464.

61. Pashin, Y. V.; Bakhitova, L. M.; Bentkhen, T. I.; Antimutagenic activity of simple phenols and its dependence on the number of hydroxyl groups. Bull Exp Biol Med. 1986, 102, 1121-1123.

62. Woodle, M. C.; Lasic, D. D.; Sterically stabilized liposomes. Biochim. Biophys. Acta 1992, 1113, 171-199.

63. Woodle, M. C.; Newman, M. S.; Cohen, J. A.; Sterically stabilized liposomes: physical and biological properties. J Drug Target 1994, 2, 397-403.

64. Woodle, M. C.; Newman, M. S.; Collins, L. R.; Efficient evaluation of long circulating or stealth liposomes by studies of in vivo blood-circulation kinetics and final organ distribution in rats. Biophys J. 1990, 57, A261.

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67. Rodríguez-Pulido, A.; Kondrachuk, A. I.; Prusty, D. K.; Gao, J.; Loi, M. A.; Herrmann, A.; Light-Triggered Sequence-Specific Cargo Release from DNA Block Copolymer–Lipid Vesicles. Angew. Chem. Int. Ed. 2013, 52, 1008-1012.

68. Chiu, H. C.; Lin, Y. W.; Huang, Y. F.; Chuang, C. K.; Chern, C. S.; Polymer Vesicles Containing Small Vesicles within Interior Aqueous Compartments and pH-Responsive Transmembrane Channels. Angew. Chem. Int. Ed. 2008, 47, 1875-1878.

69. Volodkin, D. V.; Skirtach, A. G.; Möhwald, H.; Near-IR Remote Release from Assemblies of Liposomes and Nanoparticles. Angew. Chem. Int. Ed. 2009, 48, 1807-1809.

70. Leung, S. J.; Romanowski, M.; Light-Activated Content Release from Liposomes. Theranostics 2012, 2, 1020-1036.

71. Li, L.; Hagen, ten T. L.M.; Hossann, M.; Süss, R.; Rhoon, van G. C.; Eggermont, A. M.M.; Haemmerich, D.; Koning, G. A.; Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy. J Control Release 2013, 168, 142-150.

72. Tai, L.; Tsai, P.; Wang, Y.; Wang, Y.; Lo, L.; Yang, C.; Thermosensitive liposomes entrapping iron oxide nanoparticles for controllable drugrelease. Nanotechnology 2009, 20, 1-9.

73. Nappini, S.; Bombelli, F. B.; Bonini, M.; Nord, B.; Baglioni. P.; Magnetoliposomes for controlled drug release in the presence of low-frequency magnetic field. Soft Matter 2010, 6, 154-162.

74. Wang, J.; Ayano, E.; Maitani, Y.; Kanazawa, H.; Tunable Surface Properties of Temperature-Responsive Polymer-Modified Liposomes Induce Faster Cellular Uptake, ACS Omega 2017, 2, 316-325.

75. Monnier, C. A.; Burnand, D.; Rutishauser, B. R.; Lattuada, M.; Petri-Fink, A.; Magnetoliposomes: opportunities and challenges. Eur J Nanomed. 2014, 6, 201-215.

76. Amstad, E.; Kohlbrecher, J.; Müller, E.; Schweizer, T.; Textor, M.; Reimhult, E.; Triggered Release from Liposomes through Magnetic Actuation of Iron Oxide Nanoparticle Containing Membranes. Nano Lett. 2011, 11, 1664-167.

77. Laurent, S.; Saei, A.A.; Behzadi, S.; Panahifar, A.; Mahmoudi, M.; Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: opportunities and challenges. Expert Opin Drug Deliv. 2014, 11, 1449-1470.

78. Woodle, M. C.; Surface-modified liposomes: assessment and characterization for increased stability and prolonged blood circulation. Chem. Phys. Lipids 1993, 64, 249-262.

79. Anaya, M.; Kwak, M.; Musser, A. J.; Müllen, K.; Herrmann, A.; Tunable Hydrophobicity in DNA Micelles: Design, Synthesis, and Characterization of a New Family of DNA Amphiphiles. Chem. Eur. J. 2010, 16, 12852-12859.

Chapter 2

Stability Study of Lipid-DNA on

the Liposomal Membrane

Parts of this chapter were published in: Chem. Eur. J. 2017, 23, 9391-9396.

Chapter 2

32

2.1 Introduction

Deoxyribonucleic acid (DNA) is a macro molecule that carries hereditary

information of all known living organisms and many viruses. Its double-

stranded helix structure was discovered by Watson & Crick in 1953,1 which

has greatly fueled many technologies dealing with DNA and hence

revolutionized modern science. In recent years DNA has become a valuable

functional building block and tool in nanotechnology and material science

due to the unique nature and properties of DNA and DNA hybrid materials.

A wide variety of products and applications have been realized using DNA

technologies among which is incorporating DNA with a functional group

and utilizing its information-carrying capability to develop DNA detection

systems. For instance, fluorescent dye-labeled DNA was used as probe

monitor in PCR2 or for sequence analysis.3,4 Additionally, coupling DNA

strands with moieties like polymers or nanoparticles changes the

morphological structure and introduces new functionalities, which are

Stability Study of Lipid-DNA on the Liposomal Membrane

33

different from conventional polymers. For instance, DNA conjugated gold

nanoparticles were used in DNA microarray technology.5-7 Another

functional moiety chemically conjugated with DNA consists of hydrophobic

molecules, such as long alkyl chains, cholesterol, or fatty acids, resulting in

amphiphiles, which spontaneously form nanoparticles in solution and

enhance the pharmacokinetic behavior and trans-membrane delivery.

Their amphiphilic nature arises from the hydrophilic DNA backbone

containing charged phosphodiester bonds and the hydrophobicity of

attached alkyl chains.8 These nanoparticles can be further functionalized

through hybridization of a modified complimentary DNA or internalization

of payloads in the hydrophobic core.9

Our group reported the synthesis and characterization of a family of DNA

amphiphiles containing hydrophobically modified nucleobases.10,11

Specifically, 1-dodecyne (C12H22) was attached to a uracil base which was

further attached to the 5’ or 3’ position of a DNA sequence (Fig. 2.1A). In

aqueous environment, due to their amphiphilic nature, lipid-DNA self-

assembles into micelles whereby the hydrophilic DNA strands shield the

hydrophobic lipid core. These DNA micelles can be loaded with cargo by

hydrophobic interactions or hybridization with functionalized

complementary DNA (Fig. 2.1B). The aggregation properties of lipid-DNA

can be relatively easy manipulated by changing the length of the lipid part

or the number and position of the modified uracil bases within the DNA

sequence. Fig. 2.1C shows three different lipid-DNAs. U2M and U2T are

lipid DNA with two modified uracil bases either in the middle or at the

terminus and U4T represents lipid DNA with four modified uracil bases at

the 5’ end.10

Because of the amphiphilic and sequence specific properties, lipid-DNA can

be used for liposome surface modification by insertion of the hydrophobic

part into the membrane while the hydrophilic DNA is exposed to the

aqueous medium. Compared with existing terminal modifications, our

design allows the precise and easy introduction of hydrophobic units at

arbitrary positions and numbers in a DNA sequence through conventional

solid-phase synthesis. In this chapter, DNA was modified with four lipid

Chapter 2

34

chain modified nucleobases at both terminals and it was used to anchor it

to phospholipid membranes.

Fig. 2.1 Structure of lipid modified nucleotide and representation of lipid–DNAs. (A) Chemical

structure of the lipid-modified uracil nucleobase. (B) Lipid-DNAs self-assemble to form DNA

micelles due to their amphiphilic nature. These self-assembled structures can carry cargo by

hydrophobic interaction (1) or by hybridization with a functionalized complementary DNA (2).

(C) Schematic representation of the ss and ds lipid–DNA amphiphiles (U2M, U2T, and U4T) and

their propensity to undergo Watson-Crick base pairing.10


35

2.2 Results and Discussion

2.2.1 Lipid-DNA design and characteristics

To obtain stable incorporation of DNA into the liposomal bilayer, we use

lipid-DNA (U4T-18), which has been designed to contain four modified

uracil nucleobases at the 5’ position of a 18-mer oligonucleotide (including

the 4 lipid modified uracil bases). CrU4T-18 is complementary to U4T-18

with the lipid anchor at the opposite terminus (i.e. the 3’ position). Cr-

ATTO488 is a 14-mer DNA complementary to U4T-18 and was covalently

attached an ATTO488 dye to the 3’ end (Table 2.1).

Table 2.1 Sequences of modified DNA.

Name Sequence (5’→ 3’)*

U4T-18 UUUUGCGGATTCGTCTGC

CrU4T-18 GCAGACGAATCCGCUUUU

14mer GCGGATTCGTCTGC

Cr-ATTO488 GCAGACGAATCCGC-ATTO488

*: U represents the lipid-modified uracil base.

U4T-18 can be attached to the liposome surface by insertion of four lipid-

modified nucleobases into the lipid membrane while the remaining 14mer

DNA part is protruding into the aqueous medium. This DNA unit can

hybridize with the DNA part from CrU4T-18 or Cr-ATTO488 (Fig. 2.2A).

According to the results from polyacrylamide gel electrophoresis (PAGE), a

lower electrophoretic mobility of hybridized lipid-DNA (lane 2) is observed

compared to ssDNA controls (lane 1 and lane 3), indicating successful

Watson-Crick base pairing (Fig. 2.2B).

After confirming hybridization, the melting temperature (Tm) of the ds-

lipid-DNA (U4T-18+Cr-ATTO488) was determined. The ds-lipid-DNA and

ds14mer (14mer+Cr-ATTO488) were heated at 0.5 °C/min while

measuring the absorption at 260 nm. Afterwards the first derivative of the

curve was calculated and Tm of the ds DNA was taken at maximum slope.

Chapter 2

36

The Tm value of lipid-DNA (62.5 °C) is very close to that of 14mer (63.6 °C)

(Fig 2.2C, D). The result indicates that lipid chains have little influence on

the melting temperature.

Fig. 2.2 (A) Schematic representation of U4T-18 hybridization with Cr-ATTO488 on the

surface of liposomes. (B) Native PAGE characterization of lipid-DNA (20% TBE gel, 100V,

80min). Lane 1: U4T-18, lane 2: U4T-18 + Cr-ATTO488, lane 3: Cr-ATTO488. (C) Melting curve

of dsDNA, U4T-18 + Cr-ATTO488. (D) Melting curve of dsDNA, 14mer + Cr-ATTO488. Melting

curve (black squares, left Y-axis) and calculated derivative for corresponding sample (red

circle, right Y-axis).

2.2.2 Characterization of the incorporation of lipid-DNA in liposomal

bilayer.

After synthesis of the nucleobase-modified DNA hybrids and testing their

ability for Waston-Crick base pairing, the lipid DNAs were stably anchored

into the membrane of DOPC:DOPE:cholesterol lipid vesicles, while the

oligonucleotides remained available for hybridization, as demonstrated by

a Fluorescence Resonance Energy Transfer (FRET) assay.12


37

Since ATTO488 and rhodamine dyes show energy transfer when there is a

sufficiently short distance between them,13 ATTO488 was covalently

attached to the 3’ end of a 14-mer DNA complementary to U4T-18 (Cr-

ATTO488) to act as a donor. In parallel, rhodamine-functionalized

phospholipid (Rh-DHPE) was incorporated in the liposomal bilayer to

function as an acceptor (Fig. 2.3A). To observe the dynamic emission

changes of donor and acceptor after adding Cr-ATTO488, fluorescence

emission spectra with excitation at 470 nm of Cr-ATTO488 (donor,

emission maximum 520 nm) and Rh-DHPE (acceptor, emission maximum

592 nm) were recorded over 30 min (Fig. 2.3B). The fluorescence of donor

significantly decreased by adding Cr-ATTO488 and the fluorescence of

acceptor slightly increased at the same time, illustrating that FRET is

induced by DNA hybridization.

Fig. 2.3 (A) Schematic of FRET assay demonstrating that oligonucleotides anchored into

liposomal bilayers via lipid-DNA remain available for hybridization. (B) Fluorescence emission

of Cr-ATTO488 (donor, emission maximum 520 nm) and Rh-DHPE (acceptor, emission

maximum 592 nm) followed over 30 min after adding Cr-ATTO488.

Meanwhile, as demonstrated by the increase in the maximum intensity

ratio I592/I520 (acceptor/donor peak) (Fig. 2.4D), hybridization only

occurred upon mixing of Cr-ATTO488 with U4T-18-grafted Rh-DHPE-

containing vesicles, positioning both dyes sufficiently close to each other to

achieve FRET (Fig. 2.4A), whereas for vesicles containing non-

complementary lipid-DNA, CrU4-18, (Fig. 2.4B) or no lipid-DNA at all (Fig.

2.4C), no FRET was observed.

Chapter 2

38

Fig. 2.4 Anchoring of lipid-DNA in the membrane and hybridization on the vesicle surface

leads to Fluorescence Resonance Energy Transfer (FRET) upon hybridization of donor-

modified complementary DNA with DNA-functionalized, acceptor-containing vesicles. (A)

FRET is achieved when complementary Cr-ATTO488 DNA hybridizes with U4T-18 and brings

the donor close to the acceptor, rhodamine, positioned in the membrane. If hybridization is not

possible, either due to mismatch of the two DNA strands (B) or the absence of membrane-

grafted DNA (C) FRET does not occur. (D) Fluorescence spectra of systems capable of FRET

(red) and non-FRET controls, either due to DNA mismatch (blue) or absence of membrane-

grafted DNA (green).

Disruption of vesicles by addition of Triton X-100 to a final concentration of

0.3% (v/v) resulted in a drop in FRET in the U4T-18 vesicles hybridized

with Cr-ATTO488 (Fig. 2.5A vs Fig 2.4D), confirming that FRET was

indeed caused by bringing the donor in close vicinity to the acceptor dye

located in the liposomal membrane. As expected, in two control non-FRET

systems in which DNA hybridization could not occur, either due to absence

of DNA in the membrane (Fig. 2.5B) or the presence of non-

complementary DNA (Fig. 2.5C) energy transfer from donor to acceptor


39

was prevented. Therefore, similar spectra were observed before and after

liposomal disruption.

Fig. 2.5 To further investigate the engraftment of the lipid-DNA hybrids into the membrane,

FRET liposomes were disrupted with Triton X-100 at a final concentration of 0.3 % (v/v).

Fluorescence spectra of FRET liposomes before and after adding Triton X-100 (A). Similar

spectra were observed in control experiments before and after liposomal disruption, either due

to the absence of DNA on the membrane (B) or the presence of non-complementary DNA (C).

2.2.3 Temporal stability of lipid-DNA in the liposomal membrane.

To study whether the incorporation of U4T-18 in the membrane is stable

overtime, FRET (U4T-18/Cr-ATTO488/Rh-DHPE) liposomes were

incubated with non-FRET (NF) liposomes (Fig. 2.6A) at different ratios

(1:1, 1:5 and 1:10).

Chapter 2

40

Fig. 2.6 Measurement of stability of lipid-DNA in liposomes over time. FRET (U4T-18/Cr-

ATTO488/Rh-DHPE) liposomes were incubated with non-FRET (NF) liposomes (A) at different

ratios (1:1, 1:5 and 1:10), and the relative Rh-DHPE/ATTO488 (IA/ID) emission intensity ratio

was monitored over 24 h after mixing (B). Fluorescence spectra of Cr-ATTO488/Rh-DHPE pair

in FRET liposomes mixed with NF liposomes at different ratio (v/v): 1:1(red line), 1:5(blue line),

1:10(green line) (C). Solid and dashed lines represent the spectra of the mixed systems before

and after adding Triton X-100, respectively.

The relative Rh-DHPE/ATTO488 (IA/ID) emission intensity ratio of the

three systems was monitored over 24 h after mixing (Fig. 2.6B). If lipid-

DNA redistributes from FRET liposomes to NF liposomes, a decrease in

relative fluorescence of acceptor peak would be observed. After 24 h, some

of the acceptor intensity had dropped, but the relative fluorescence IA/ID of

the mixture remained at a similar value as that during the initial

measurement before non-FRET liposomes were added. The results

demonstrate that the lipid–DNA is stably anchored in the liposomes over at

last 24 hours. Fig. 2.6C shows the fluorescence spectra of Cr-ATTO488/Rh-

DHPE pair in FRET liposomes mixed with NF liposomes at different ratio

before and after liposomal disruption.


41

Moreover, lipids were mixed with U4T-18 at different molar ratios (5000,

1000, 100, 62.5). The final concentration of Cr-ATTO488 and lipid-mixture

(DOPC+DOPE) were kept at 7.32 µM and 0.45 mM, respectively, in all FRET

experiments. The results show the I592/I520 ratio increased markedly with

higher U4T-18 densities in the membrane (Fig. 2.7, Table 2.2). These

results demonstrate that when more lipid DNA is incorporated into the

membrane more DNA strands can be attached to this vesicle surface by

hybridization (Table 2.2).

Fig. 2.7 U4T-18/Rh-DHPE fluorescence spectra of FRET liposomes mixed with Cr-ATTO488 at

different lipid/U4T-18 ratios. The inset shows a zoom-in of the acceptor Rh-DHPE peak. Solid

lines and dashed lines represent the spectra of the FRET system before and after adding Triton

X-100, respectively. Lipids were mixed with U4T-18 at different molar ratios (5000, 1000, 100,

62.5).

Table 2.2 The acceptor/donor fluorescence intensity ratios (I592/I520) at different lipid/U4T-18

ratios.

Lipid : U4T-18

ratio

U4T-18:liposome

ratio I592/I520 FRET system

5000 8 0.22

1000 38 0.24

100 380 0.31

62.5 608 0.44

Chapter 2

42

2.3 Conclusion

In conclusion, we proposed a powerful new approach employing lipid-DNA

which contains four lipid chains modified nucleobases to tightly anchor the

nucleotide to the lipid membrane. The incorporation and stability of lipid-

DNA on the liposomal membrane were proved by FRET. FRET was

achieved when the hybridization occurred between Cr-ATTO488 and U4T-

18, which brought the donor (Cr-ATTO488) close to the acceptor

(rhodamine) that was positioned in a U4T-18 functionalized membrane.

Meanwhile, the I592/I520 (acceptor/donor peak) ratio increased markedly

with higher U4T-18 densities in the membrane, and disruption of vesicles

by addition of Triton X-100 resulted in a drop of FRET vesicles system,

confirming that FRET was indeed caused by bringing the donor in close

vicinity to the acceptor dye located in the liposomal membrane. Finally, the

lipid–DNA remained stably anchored in the liposomes for at least 24 hours.

2.4 Experimental Section

2.4.1 Materials

Cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)

and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased

from Avanti Polar Lipids (Alabaster, USA) (Fig 2.8A-C, purity >99%) and

used without further purification. Headgroup-labeled phospholipid,

Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-

phosphoethanolamine (triethylammonium salt) (Rh-DHPE) was purchased

from Invitrogen (Amsterdam, Netherlands), and used as received (Fig.

2.8D). The DNA-dye conjugate Cr-ATTO488 was purchased from

Biomers.net GmbH (Ulm, Germany). Trition X-100 (10% in water), and

Tris/HCl buffer were purchased from Sigma-Aldrich (St. Louis, United

States). Anhydrous CHCl3 was purchased from Acros Organics (Geel,

Belgium) and stored over molecular sieves. For all experiments, ultrapure

water (specific resistance > 18.4 MΩ cm) was obtained by a Milli-Q water

purification system (Sartorius).


43

Fig. 2.8 Structures of lipids: (A) DOPC, (B) DOPE, (C) Cholesterol; fluorescent lipids: (D) Rh-

DHPE.

2.4.2 Synthesis and characterization of amphiphilic oligonucleotides

The synthesis of 5-(dode-1-cynyl) deoxyuracil and 5-(dode-1-cynyl)

deoxyuracil phosphoramidite were reported previously (Fig. 2.9).10,11 In

short, the modified uracil phosphoramidite was dissolved in CH3CN to a

final concentration of 0.15 M in the presence of 3 Å molecular sieves and

the prepared solution was directly connected to a DNA synthesizer (ÄKTA

oligopilot plus, GE Healthcare (Uppsala, Sweden)). Oligonucleotides were

synthesized on a 10 μmol scale using standard β-cyanoethylphosphoramidi

-te coupling chemistry. Deprotection and cleavage from the PS support was

carried out by incubation in concentrated aqueous ammonium hydroxide

solution for 5 h at 55 °C. Following deprotection, the oligonucleotides were

purified by using reverse-phase chromatography, using a C15 RESOURCE

RPCTM 3 mL reverse phase column (GE Healthcare) through a custom

gradient elution (A: 100 mM triethylammonium acetate (TEAAc) and 2.5%

acetonitrile, B: 100 mM TEAAc and 65% acetonitrile). Fractions were

Chapter 2

44

desalted using centrifugal dialysis membranes (MWCO 3000, Sartorius

Stedim). Oligonucleotide concentrations were determined by UV

absorbance using extinction coefficients. Finally, the identity and purity of

the oligonucleotides was confirmed by RPC-HPLC (Fig. 2.10) and MALDI-

TOF mass spectrometry (Fig. 2.11).

Fig. 2.9 Synthesis of 5-(dode-1-cynyl) deoxyuracil 2 and 5-(dode-1-cynyl) deoxyuracil

phosphoramidite 3.

Fig. 2.10 MALDI-TOF mass spectra of lipid-DNAs. (A) U4T-18, (B) CU4T-18 and (C) CrU4T-18.


45

Fig. 2.11 RPC HPLC analysis of purified lipid-DNAs: (A) U4T-18, (B) CU4T-18 and (C) CrU4T-18.

Numbers beside the elution peaks represent the buffer B contents when lipid-DNAs were eluted.

2.4.3 Preparation and characterization lipid-DNA liposomes

An appropriate amount of freeze-dried lipid-DNA was mixed with

DOPC:DOPE:Cholesterol (50:25:25 mol% in chloroform), to obtain the

required lipid:lipid-DNA ratio. Afterwards, chloroform was removed by

evaporation under an air stream and then under vacuum overnight. An

aqueous buffer (100 mM NaCl, 20 mM Tris, pH 7.5) was added to the flask

and the solution was vortexed and freeze-thawed 5 times. Subsequently,

the dispersion was extruded 21 times, using an extruder and 100 nm

polycarbonate membranes (Whatman), to obtain unilamellar vesicles. After

extrusion, external buffers of each sample were removed by size exclusion

chromatography. The column was filled with Sephadex G-75 (GE

Healthcare Life Sciences) and equilibrated with buffer (100 mM NaCl, 20

mM Tris, pH 7.5). Lipid-DNA liposomes were used within one day. All

liposomal formulations had an average diameter of around 130 nm as

determined by DLS (ALV/CGS-3 ALV-Laser Vertriebsgesellschaft mbH,

Langen, Germany). The ratio between lipid and U4T-18 was 500:1, unless

stated otherwise.

Chapter 2

46

2.4.4 Calculation of lipid-DNA/liposome ratio.

The amount of lipid-DNAs per liposome was calculated using the equation:

where Φ is the number of lipids per liposome which can be calculated from

geometrical considerations:

where Souter and Sinner are the outer and inner surface area of the spherical

liposomes. Assuming the thickness of the lipid bilayer is 5 nm.14,15 α is the

average cross-sectional area of the lipid headgroups, which is assumed to

be (2*80+65)/3=75 Å for DOPC:DOPE(2:1 molar ratio).16 Router is the

averaged radius of spherical liposomes, which was determined by DLS.

2.4.5 Characterization of lipid-DNA incorporation in liposomes

measured by Fluorescence Resonance Energy Transfer (FRET) assay

Fluorescence emission spectra of Cr-ATTO488 (donor) and Rh-DHPE

(acceptor) in the 500–700 nm region were recorded with excitation at 470

nm using a SPECTRAMAX M2 (Molecular Devices) fluorescence

spectrophotometer. Measurements were carried out at constant

temperature of 25.0 °C, using a 100 mM NaCl, 20 mM Tris, pH 7.5 buffer.

2.4.6 FRET assay via DNA hybridization

U4T-18 was incorporated in Rh-DHPE/(DOPC+DOPE) (3:97 molar ratio)

liposomes to obtain U4T-18 liposomes with a lipid to U4T-18 ratio of 500:1.

Subsequently, an aliquot of these liposomes was mixed with a small

amount of Cr-ATTO488 such that [U4T-18] = [Cr-ATTO488] = 0.906 μM

and with a final lipid (DOPC+DOPE) concentration of 0.45 mM. Then, U4T-

18 and Cr-ATTO488 were hybridized using an Eppendorf Mastercycler

(Germany). The protocol consisted of heating the mixture 15 min to 40 °C


47

and slowly cooling to 4 °C over a period of 140 min. Afterwards, the

emission spectra of Cr-ATTO488/Rh-DHPE pair were measured.

Author contributions

Meng Z designed and conducted the experiments, performed data analysis

and wrote the manuscript. Liu Q and de Vries JW synthesized lipid-DNA.

Herrmann A supervised the project.

Chapter 2

48

References

1. Watson, J. D.; Crick, F. H.; Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 1953, 171, 737-738.

2. Kapandis, A. N.; Weiss, S.; Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J. Chem. Phys. 2002, 117, 10953-10964.

3. Demidov, V. V.; PNA and LNA throw light on DNA. TRENDS in Biotechnology 2003, 4-7.

4. Liu, Z.; Liu, B.; Ding, J.; Liu. J.; Fluorescent sensors using DNA-functionalized graphene oxide. Anal Bioanal Chem 2014, 406, 6885-6902.

5. Cho, H.; Jung, J.; Chung, B. H.; Scanometric analysis of DNA microarrays using DNA intercalator-conjugated gold nanoparticle. Chem. Commun. 2012, 48, 7601-7603.

6. Niemeyer, C. M.; Ceyhan, B.; Noyong, M.; Simon, U.; Bifunctional DNA–gold nanoparticle conjugates as building blocks for the self-assembly of cross-linked particle layers. Biochem Biophys Res Commun. 2003, 311, 995-999.

7. Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A.; DNA-programmable nanoparticle crystallization. Nature 2008, 451, 553-556.

8. Kwak M.; Herrmann, A.; Nucleic acid amphiphiles: synthesis and self-assembled nanostructures. Chem. Soc. Rev. 2011,40, 5745-5755.

9. Kwak, M.; Musser, A. J.; Lee, J.; Herrmann, A.; DNA-functionalised blend micelles: mix and fix polymeric hybrid nanostructures. Chem. Commun. 2008, 29, 326.

10. Anaya, M.; Kwak, M.; Musser, A. J.; Müllen, K.; Herrmann, A.; Tunable Hydrophobicity in DNA Micelles: Design, Synthesis, and Characterization of a New Family of DNA Amphiphiles. Chem. Eur. J. 2010, 16, 12852-12859.

11. Kwak, M.; Minten, I. J.; Anaya, D. M.; Musser, A. J.; Brasch, M.; Nolte, R. J. M.; Müllen, K.; Cornelissen, J. J. L. M.; Herrmann, A.;Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers. J. Am. Chem. Soc. 2010, 132, 7834-7835.


13. Alfonta, L.; Singh, A. K.; Willner, I.; Liposomes Labeled with Biotin and Horseradish Peroxidase: A Probe for the Enhanced Amplification of Antigen-Antibody or Oligonucleotide-DNA Sensing Processes by the Precipitation of an Insoluble Product on Electrodes. Anal. Chem. 2001, 73, 91-102.

14. Leonenko, Z. V.; Finot, E.; Ma, H.; Dahms, T. E. S.; Cramb, D. T.; Investigation of Temperature-Induced Phase Transitions in DOPC and DPPC Phospholipid Bilayers Using Temperature-Controlled Scanning Force Microscopy. Biophys J. 2004, 86, 3783-3793.


49

15. Gramse, G.; Perez, A. D.; Edwards, M. A.; Fumagalli, L.; Gomila, G.; Nanoscale Measurement of the Dielectric Constant of Supported Lipid Bilayers in Aqueous Solutions with Electrostatic Force Microscopy. Biophys J. 2013, 104, 1257-1262.

16. Wiethoff, C. M.;Gill, M. L.; Koe, G. S.; Koe, J. G.; Middaugh, C. R.; The Structural Organization of Cationic Lipid-DNA Complexes. J. Biol. Chem. 2002, 277, 44980-44987.

Chapter 3

Efficient Fusion of Liposomes by Nucleobase

Quadruple-Anchored DNA

Parts of this chapter were published in: Chem. Eur. J. 2017, 23, 9391-9396.

Chapter 3

52

3.1 Introduction

Liposomes are a particularly effective class of nanocontainers, being able to

encapsulate and protect both small molecules and bio-macromolecules,

such as proteins or DNA.1-3 The engineering of liposomes has advanced to a

level that enables the manipulation of their surfaces with specific ligands in

order to improve their functionality. For instance, proteins, carbohydrates

and vitamins have been used as targeting units to improve the cellular

specificity of these nanocontainers. Moreover, some “smart” vesicle designs

allow the release of the encapsulated cargo through physicochemical

responses of the liposomal membrane to external stimuli4,5 or by

incorporation of transport channels.6-9 Another strategy by which

liposomes can deliver their payload to cells, is via membrane fusion,10-12

which has previously been demonstrated for drug13-16 and gene delivery17-

20 applications.

In many cellular processes, including exocytosis, endocytosis, and the

transfer of membrane proteins between cellular compartments, membrane

fusion plays a crucial role.21,22 Most membrane fusion events follow a

similar order：docking, hemifusion and full fusion. As part of the docking

process, membranes are brought into close proximity, which can cause the

outer layers to merge while the inner layers stay separated, resulting in

hemifusion. Full fusion is achieved when the outside and inside layers of

both membranes merge and content mixing occurs. Recently, several

Efficient Fusion of Liposomes by Nucleobase Quadruple-Anchored DNA

53

groups have reported hemifusion and full fusion of liposomes by exploiting

Watson-Crick base pairing of complementary membrane-anchored

oligonucleotides. In these studies, DNA was grafted onto the liposomal

surface using cholesterol- or fatty acid-derivatives conjugated at the 5’- or

3’-end of the DNA oligomers.23-26 However, full fusion induced by these

systems was only achieved to a limited extent, i.e. below 4%,25,27 or with a

significant degree of content leakage.28 These limitations may be related to

DNA duplex formation and/or linkers separating the two membrane

surfaces, thereby inhibiting further membrane contact and preventing full

fusion. However, the design of the hydrophobic anchor employed to graft

the DNA into the lipid membrane could play a crucial role as well. Once two

vesicles are brought close enough for full fusion, insufficient affinity of the

hydrophobic domain of the DNA-conjugate for the bilayer or weak

mechanical coupling between the anchor and the oligonucleotides may

disable further fusion (Fig. 3.1A).

Here, we report of a powerful new approach for anchoring DNA on a

membrane and to achieve vesicle-vesicle fusion by employing DNA that is

modified with lipid chains at four nucleobases (Fig. 3.1B, C). This strategy

achieved a highly stable incorporation of DNA into the liposomal bilayer,

thereby limiting dissociation and keeping the base-pairing nucleotides

close to the surface and allowing for a markedly more efficient full fusion as

compared to other, previously reported, anchoring strategies.

Fig. 3.1 Schematic representation of vesicle fusion using lipid-modified oligonucleotides. An

oligonucleotide anchored with a single unit might be pulled out of the membrane after

hybridization and aggregation of two vesicles, which hinders full fusion (A). In the strategy

presented here, highly efficient vesicle fusion was induced by DNAs that were modified at the

nucleobases, enabling stable grafting of quadruple anchored oligonucleotides capable of non-

zipper-oriented (B) and zipper-oriented hybridization of complementary strands (C).

Chapter 3

54


Table 3.1. Sequences of DNA modified with lipid-nucleobases, poly(propylene oxide) and

cholesterol.

Name Sequence (5’→ 3’)*

U4T-18 UUUUGCGGATTCGTCTGC

CU4T-18 UUUUGCAGACGAATCCGC

CrU4T-18 GCAGACGAATCCGCUUUU

Cr-ATTO488 GCAGACGAATCCGC-ATTO488

U2T-16 UUGCGGATTCGTCTGC

CrU2T-16 GCAGACGAATCCGCUU

22PPO poly(propylene oxide)-5'-CCTCGCTCTGCTAATCCTGTTA-3'

Cr22PPO 5'-TAACAGGATTAGCAGAGCGAGG-3'-poly(propylene oxide)

14Chol Cholesterol-5'-GCGGATTCGTCTGC-3'

Cr14Chol 5'-GCAGACGAATCCGC-3'-Cholesterol

*: U represents the lipid-modified uracil base.

In the approach hereto achieve fusion employing novel anchoring units,

complementary oligonucleotides containing four uracil (U) bases modified

with dodec-1-yne (C12H22) at 3’ or 5’ position of DNA oligomers were

employed29: enabled by the previously published phosphoramidite

building block and automated DNA synthesis, U4T-18 has been fabricated

to contain four modified uracil nucleobases at the 5’ position of the 18-mer

oligonucleotide (Table 3.1), whereas CU4T-18 is complementary to U4T-

18 with the lipid anchor at the same terminus (i.e. the 5’ position) as U4T-

18.

Upon hybridization, the lipid functionalities are oriented in the DNA double

helix in a so-called ‘non-zipper’-like arrangement (Fig. 3.2). In contrast,

CrU4T-18, which is also complementary to U4T-18, was prepared with the


55

lipid anchor on the opposite terminus (i.e. the 3’ position) and therefore

allows for a ‘zipper’-like orientated hybridization.

Fig. 3.2 Schematic representations of lipid-modified DNA hybrids in non-zipper and zipper like

arrangements.

3.2.1 Docking of Liposomes Grafted with Quadruple-Anchored DNA

After establishing that lipid-modified oligonucleotides remained stably

incorporated into phospholipid bilayers for extended period of times, their

functionality for hybridization-induced vesicle-vesicle interaction was

explored. The fusion of lipid bilayers is a three-step process: docking,

hemifusion and full fusion. DNA hybridization allows docking of vesicles by

overcoming the repulsive hydration forces between the lipid-headgroups,

i.e. bringing the lipid bilayers of the liposomes functionalized with

complementary DNA into close proximity to each other. Liposomal docking

was observed when U4T-18 vesicles were incubated in a 1:1 ratio with

vesicles decorated with the complementary DNA sequence (CrU4T-18 or

CU4T-18), each formulation with an average diameter of around 130 nm.

After 5 hours, the average liposomal diameter, as determined by dynamic

light scattering (DLS), increased from 130 nm to around 350 nm and 300

nm, for the zipper and non-zipper orientated hybridization, respectively,

while the diameter of the U4T-18 vesicles alone did not change notably

(Fig. 3.3). This indicates that DNA hybridization and vesicle aggregation

Chapter 3

56

has taken place in both binding modes, although zipper orientation

hybridization resulted in on average slightly larger objects.

Fig. 3.3 Time evolution of average diameter measured by DLS of vesicles functionalized with

DNA. Upon incubation of U4T-18-grafted vesicles (diameter 130 nm) with vesicles of equal size

containing complementary DNA sequences, hybridization in either zipper (CrU4T-18, red) or

non-zipper (CU4T-18, blue) orientation, resulted in an increase in average diameter of the

entire population. For U4T-18-grafted vesicles alone (green), the average diameter remained

constant.

The docking of U4T-18 liposomes was also investigated with cryogenic

transmission electron microscopy (cryo-TEM), and no apparent

aggregation was observed in the absence of complementary DNA-

functionalized liposomes (Fig. 3.4A). In contrast, strong aggregation was

observed in the mixture of U4T-18 and CU4T-18 decorated liposomes

when incubated overnight (Fig. 3.4B), as well as in the mixture of U4T-18

and CrU4T-18 decorated liposomes (Fig. 3.4C, 3.4D). Moreover, signs of

liposomal fusion were present in the U4T-18/CrU4T-18 zipper-like

arrangement sample, such as bridging membranes and the presence of

large vesicles (red circles, Fig. 3.4D). The molar ratio between

phospholipids and lipid-DNA was optimized to be 500:1 (around 140 DNA

strands per vesicle, data not shown), unless stated otherwise.


57

Fig. 3.4 Cryo-TEM images of (A) U4T-18 decorated liposomes, (B) a mixture of U4T-18 and

CU4T-18 decorated liposomes, and (C, D) a mixture of U4T-18 and CrU4T-18 decorated

liposomes. The red circles in (D) indicate vesicles that are suggestive of hemifusion. (All the

samples were incubated at 4 °C overnight.)

3.2.2 Hemifusion of Liposomes Grafted with Quadruple-Anchored DNA

To investigate the second step of vesicle fusion, i.e. hemifusion, a lipid

mixing assay based on FRET was conducted.30 Similar to a procedure

reported previously,31 the membranes of liposomes decorated with U4T-18

were stained with 0.5 mol% NBD-DHPE (donor) and 0.5 mol% Rh-DHPE

(acceptor) (FRET liposomes), while complementary DNA-functionalized

vesicles, grafted with CrU4T-18 or CU4T-18, were prepared without

fluorescently-labeled lipids (non-fluorescent liposomes). Lipid mixing

between FRET and non-fluorescent liposomes would increase the average

distance between donor and acceptor dyes, thereby attenuating FRET and

consequently increasing donor emission. Both zipper orientated and non-

zipper orientated hybridization were able to induce lipid mixing to a

similar exent (± 40%, Fig. 3.5), suggesting that hemifusion occurs

irrespective of the orientation of DNA hybridization.

Chapter 3

58

Fig. 3.5 Lipid mixing between U4T-18 grafted vesicles loaded with 0.5 mol% NBD-DHPE and

0.5 mol% Rh-DHPE and CrU4T-18 (zipper, red) or CU4T-18 (non-zipper, blue) decorated

vesicles measured by an increase in NBD emission due to a reduction in FRET efficiency. For

NBD/Rh loaded vesicles incubated with unloaded vesicles that contained non-complementary

DNA (U4T-18), no reduction in FRET efficiency was observed (green). The NBD emission of

vesicles prepared with 0.25 mol% of NBD-DHPE and 0.25% Rh-DHPE was considered full

(100%) lipid mixing (These data represent the average of three experiments).

3.2.3 Full Fusion of Liposomes Grafted with Quadruple-Anchored DNA

The concluding step of vesicle fusion consists of content mixing, i.e. the

merging of the aqueous compartments of both liposomes. This process was

evaluated by a content mixing assay, employing a protocol as reported

previously.31 In short, the fluorescent dye sulforhodamine B was

encapsulated at a self-quenching concentration (10 mM) into U4T-18

functionalized liposomes, while CrU4T-18 or CU4T-18 functionalized

liposomes were prepared without any dye. Full fusion of the U4T-18 vesicle

with its complementary counterpart would lead to content mixing and

Sulforhodamine B dilution, thereby dequenching its fluorescence resulting

in an increase in emission.

Upon exposure of U4T-18-decorated Sulforhodamine B-containing

liposomes to complementary DNA-decorated unloaded liposomes, there

was a prominent increase of sulforhodamine B emission. The mixing

induced by DNA hybridization in the zipper orientation was markedly

higher (29%, after 1 hour) than that by DNA hybridized in non-zipper


59

orientation (18%) (Fig. 3.6), while for liposomes grafted with the same,

and therefore non-complementary, U4T-18 lipid-DNA, only a negligible

amount of dequenching occurred (2%).

Fig. 3.6 Content mixing between liposomes decorated with U4T-18 and loaded with

sulforhodamine B and unloaded liposomes functionalized with CrU4T-18 (zipper, red) or

CU4T-18 (non-zipper, blue). Content mixing was measured as an increase in sulforhodamine B

emission due to dequenching, suggesting DNA-induced full fusion. U4T-18-grafted

sulforhodamine B-loaded liposomes mixed with unloaded U4T-18 decorated liposomes, which

could not hybridize, were used as a control (green). The fluorescence intensity upon maximal

dequenching of sulforhodamine B by disruption of liposomes in 0.3% (w/v) Triton X-100 was

considered 100% content mixing (These data represent the average of three experiments).

3.2.4 Leakage Test

Leakage of the aqueous content of vesicles into the surrounding medium

during the fusion process, possibly due to pore formation, has shown to be

a significant hurdle in DNA-induced vesicle fusion.28 To distinguish clean

fusion from leaky fusion in the dye dequenching-based content mixing

assay employed here, U4T-18-grafted vesicles incubated with either CU4-

18- or CrU4T-18-grafted vesicles were precipitated using an

ultracentrifuge and the fluorescence intensity of the supernatants analyzed.

Supernatants of liposomes fused in either orientation, as well as that of

U4T-18 before fusion, displayed a very similar fluorescent intensity (Fig.

3.7), demonstrating that full fusion was achieved with minimal leakage.

The leakage was calculated to be below 2% for both DNA configurations.

Chapter 3

60

Fig. 3.7 Investigation of leaching of content after 1 hour fusion by measuring fluorescence

spectra of the incubated DNA-functionalized vesicles. Before centrifugation (A) differences in

fluorescent intensity of sulforhodamine B-loaded U4T-18 liposomes incubated with either

unloaded CrU4T-18 liposomes (red line) or unloaded CU4T-18 liposomes (blue), as compared

to sulforhodamine B-loaded U4T-18 liposomes alone (green), suggests vesicle fusion due to

dequenching of the fluorescent dye. In case vesicle fusion is accompanied by content leakage

(leaky fusion), the fluorescence intensity of the supernatants of the fusing vesicles would be

higher than that of control, non-fusing vesicles. The very similar fluorescence intensities of the

supernatants of each sample, including control, upon ultracentrifugation at 80.000g (B)

confirmed that dequenching occurred within the vesicles as a result of clean fusion, rather

than leakage of the contents into the aqueous environment.


61

3.2.5 Influence of Number of Anchoring Units on Efficacy of DNA-

Induced Full Fusion

To evaluate whether the strategy by which the DNA is anchored into the

lipid bilayer, and specifically the number of anchoring units, is a

determining factor in hybridization-induced vesicle fusion, double

anchored variants of U4T-18 comprising the same (complementary)

sequence for hybridization, but modified with only two, rather than four,

lipid-modified uracil nucleobases (U2T-16, CrU2T-16, Table 3.1), were

synthesized and evaluated. As compared to the quadruple-anchored DNAs,

incubation of vesicles functionalized with complementary U2T-16

oligonucleotides resulted in markedly lower full fusion efficacy (8%, Fig.

3.8).

Fig. 3.8 Content mixing between liposomes decorated with U2T-16 and loaded with

sulforhodamine B and unloaded liposomes functionalized with CrU2T-16. Content mixing was

measured as an increase in sulforhodamine B emission due to dequenching (red), indicating

full fusion induced by zipper-oriented hybridization. U2T-16-grafted sulforhodamine B-loaded

liposomes mixed with unloaded U2T-16 decorated liposomes, which could not hybridize, were

used as a control (green). The fluorescence intensity upon maximal dequenching of

sulforhodamine B by disruption of liposomes in 0.3% (w/v) Triton X-100 was considered 100%

content mixing (These data represent the average of three experiments).

Moreover, for vesicles that contained single anchored oligonucleotides, that

consisted of single-stranded DNA modified with poly(propylene oxide)

Chapter 3

62

(PPO)32 and cholesterol28 anchors at either terminus (Fig. 3.9 and Table

3.1), full fusion was only achieved to a moderate degree (5%, Fig. 3.10).

Fig. 3.9 Illustration of modified DNA. Chemical structure of (A) PPO-DNA and (B) Chol-DNA. (C)

Schematic representation of ss, ds PPO-DNA and Chol-DNA.


63

Fig. 3.10 Content mixing between liposomes decorated with 22PPO/Cr22PPO (A) and

14Chol/Cr14Chol (B). Content mixing was measured by increase in sulforhodamine B emission

due to dequenching (These data represent the average of three experiments).

3.3 Conclusion

These results demonstrate that, besides zipper or non-zipper orientation of

hybridization, the extent of full fusion in DNA hybridization-induced vesicle

fusion is highly dependent on the anchoring strategy of the hybridizing

nucleotides. Previously, other research groups have studied vesicle fusion

using lipid-anchored DNA. Höök et al. were the first to exploit the unique

properties of polynucleotides to induce controllable vesicle fusion via

complementary hybridization.23 In their approach, sticky-ended, double-

stranded DNA constructs were used, which were grafted into the liposomal

bilayer by means of two cholesterol anchors, each conjugated via a PEG-

linker to the termini of the double-stranded DNA anchors.33 The double-

stranded, bivalent cholesterol-anchored DNA was much more efficient in

inducing vesicle fusion than single-stranded, monovalent cholesterol-

anchored DNA, which only resulted in around 5% content mixing after 1

hour, indicating insufficient grafting stability of a monovalent anchor to

withstand the strain during DNA hybridization and bilayer reorganization.

Bivalent single-stranded oligonucleotides, i.e. two cholesterol moieties

conjugated to a single DNA, were evaluated as well,28 and although only the

efficiency regarding hemifusion, rather than full fusion, was reported,

hemifusion of vesicles grafted with complementary single-stranded,

Chapter 3

64

bivalent cholesterol-anchored DNA was similarly effective as that of their

bivalent double-stranded counterparts.

A second DNA-mediated vesicle fusion strategy, reported by Boxer et al.;

also utilized double anchored oligonucleotides. Single-stranded

complementary DNA modified with a C18 diglyceride at either terminus

was used,24 which, besides a longer chain length, are structurally relatively

similar to the U2T-16 lipid-DNAs used in the current study. The hemifusion

of vesicles functionalized with complementary diglyceride-anchored DNA

was highly efficient, illustrated by lipid mixing ratios of up to 80%,

depending on number of DNAs per vesicle24 and the presence and length of

non-hybridizing, linking sequences.25 Remarkably, however, full fusion of

vesicles grafted with the double anchored diglyceride-modified DNA

remained quite limited, with content mixing of around 2-3% for non-

repeating DNA sequences.24,25 Also taking into account the markedly

reduced full fusion achieved with the double anchored U2T-modified DNAs

as compared to the quadruple-anchored U4T-modified oligonucleotides, it

is conceivable that the number of anchoring moieties, is an important

factor in the design of lipid-DNAs and that a multivalent anchor is an

important prerequisite for efficient vesicle fusion.

Variations in experimental setup commonly obscure any comparison of

results produced in different studies, in particular of those performed in

different research groups. In order to bring the results of the current study

into context with previously reported data, cholesterol-anchored DNAs

used by Höök et al. were synthesized and evaluated in vesicles using the

content mixing assay that was also used for the U4T-18-grafted vesicles.28

Upon obtaining an extent of full fusion that was quite similar to that

reported previously by Höök et al. (Fig. 3.10B), it could be concluded that

U4T-anchored DNA indeed possesses highly favorable fusogenic properties

when incorporated into liposomal membranes, and that its remarkable

efficiency was not merely related to experimental factors.

In this study, we have established a new anchoring strategy for

oligonucleotides in vesicle membranes enabled by attaching a hydrophobic

unit to the nucleobase. The membrane anchors are incorporated into the


65

oligonucleotide by automated solid phase synthesis allowing precise

control over the position and number of hydrophobic units within a DNA

sequence. Therewith, this strategy overcomes structural limitations in the

context of terminal labeling with lipid moieties. With a zipper configuration

and four anchoring units close to 30% full fusion was achieved, which

might be related to the higher affinity of a quadruple lipid anchor to the

membrane, as compared to a double or single lipid anchor. We speculate

that strong anchoring limits (partial) dissociation during fusion, thereby

preventing leakage due to pore formation, keeping the double-stranded

DNA close to the vesicle surface, and consequently bringing docked vesicles

in close proximity to enhance full fusion. This ‘proximity effect’ is further

supported by the observation that zipper-orientated hybridization is more

efficient than non-zipper-orientated hybridization. In addition, a

conformational change of the lipid-modified DNA during hybridization

could induce a reorientation of the lipid anchors, disrupting the

arrangement of lipids around the lipid-modified nucleobases, and thereby

facilitating membrane fusion.

In the future, we will investigate DNA sequences with nucleobase mediated

anchoring of different designs, such as multiple anchoring regions within a

single strand, allowing to further improve the efficacy of the DNA-induced

vesicle fusion.


3.4.1 Materials



from Avanti Polar Lipids (Alabaster, USA) (purity >99%) and used without

further purification. The DNA-dye conjugate Cr-ATTO488 was purchased

from Biomers.net GmbH (Ulm, Germany). Trition X-100 (10% in water),

Sulforhodamin B and Tris/HCl buffer were purchased from Sigma-Aldrich

(St. Louis, United States). Anhydrous CHCl3 was purchased from Acros

Organics (Geel, Belgium) and stored over molecular sieves. Preparation of

Chapter 3

66

liposomes was performed in double deionized water (Super Q Millipore

system).


An appropriate amount of freeze-dried lipid-DNA was mixed with

DOPC:DOPE:Cholesterol (50:25:25 mol% in chloroform), to obtain the

required lipid:lipid-DNA ratio. For lipid mixing experiments, 0.5 mol%

NBD-DHPE and 0.5 mol% Rh-DHPE were included. Afterwards, chloroform

was removed by evaporation under an air stream and then under vacuum

overnight. An aqueous buffer (100 mM NaCl, 20 mM Tris, pH 7.5) was

added to the flask and the solution was vortexed and freeze-thawed 5 times.

10 mM sulforhodamine B was encapsulated in U4T-18 decorated liposomes

for content mixing. Subsequently, the dispersion was extruded 21 times,

using an extruder and 100 nm polycarbonate membranes (Whatman), to

obtain unilamellar vesicles. After extrusion, external buffers of each sample

were removed by size exclusion chromatography. The column was filled

with Sephadex G-75 (GE Healthcare Life Sciences) and equilibrated with

buffer (100 mM NaCl, 20 mM Tris, pH 7.5). Lipid-DNA liposomes were used

within one day. All liposomal formulations had an average diameter of

around 130 nm as determined by DLS (ALV/CGS-3 ALV-Laser

Vertriebsgesellschaft mbH, Langen, Germany). The ratio between lipid and

U4T-18 was 500:1, unless stated otherwise.

3.4.3 Cryo TEM

Liposomes (total lipid concentration 2 mg/mL) were deposited on a glow-

discharged holey carbon-coated grid (Quantifoil 3.5/1, QUANTIFOIL Micro

Tools GmbH). The excess of solution was blotted off with a filter paper. The

grid was vitrified in liquid ethane using a Vitrobot (FEI) and stored in liquid

nitrogen before being transferred to a Philips CM 120 cryo-electron

microscope equipped with a Gatan model 626 cryo-stage, operating at 120

kV. Images were taken in low-dose mode using slow-scan CCD camera.

3.4.4 Lipid mixing

Fluorescence measurements were performed on a Tecan Plate Reader

Infinite M1000 (Männedorf, Switzerland). NBD emission was measured


67

continuously, at 530 nm for 3500 s, upon mixing fluorescent U4T-18

decorated liposomes with non-fluorescent CU4T-18 or CrU4T-18 decorated

liposomes. The 0% value (F0) was determined by measuring NBD emission

of U4T-18 decorated liposomes, which were added to an equal volume of

U4T-18 decorated liposomes at t=0. The 100% value of lipid mixing (F100%)

was determined by measuring NBD emission of liposomes which contained

0.25mol% NBD-DHPE and 0.25% Rh-DHPE. The percentage of lipid mixing

was determined by the fluorescence (NBD) increase, %F(t). %F(t)=(F(t)-

F0)/(F100%-F0) where F(t) is the fluorescence intensity of NBD measured at

time t.

3.4.5 Content mixing

10 mM sulforhodamine B was encapsulated into liposomes decorated with

U4T-18. CU4T-18 or CrU4T-18 was grafted onto non-fluorescent liposomes.

Liposomes with encapsulated sulforhodamine B were separated from non-

encapsulated dye using Sephadex G-75 size exclusion columns equilibrated

with 100 mM NaCl, 20 mM Tris buffer, pH 7.5. After mixing two liposome

formulations, the percentage of content mixing was determined by the

increase in emission of the sulforhodamine B, %F(t)=(F(t)-F0)/(F100%-F0)

where F(t) is the fluorescence intensity of sulforhodamine B measured at

time t. The fluorescence intensity at 580 nm was monitored in a continuous

fashion for 3600 s. Measurements were performed on a Tecan Plate Reader

Infinite M1000 (Männedorf, Switzerland) at room temperature. F0 was the

fluorescence intensity measured at the time when two liposome

populations were mixed together. The 100% value (F100%) was the

fluorescence intensity measured after disruption of liposomes in 0.3%

(w/v) Triton X-100 to obtain 100% release. The fluorescence intensity of

U4T-18 decorated Sulforhodamine B liposomes mixed with U4T-18

decorated non-fluorescent liposomes was used as a negative control.

3.4.6 Evaluation of fusion-induced leakage

U4T-18-decorated liposomes loaded with sulforhodamine B, unloaded

CrU4T-18- and CU4T-18-grafted liposomes were prepared as described

above, and incubated for 1h. The dispersions were subsequently

centrifuged during 20 min at 80.000 g, at 4 °C, using OptimaTM TLX

Chapter 3

68

Ultracentrifuge (Beckman Coulter) to precipitate the liposomes.

Fluorescence emission spectra of supernatants, before and after

centrifugation in the 540–640 nm region were recorded with excitation at

520 nm using a SPECTRA max M2 (Molecular Devices) fluorescence

spectrophotometer. Measurements were carried out at constant

temperature of 25 °C.


Meng Z designed and conducted the experiments, performed data analysis

and wrote the paper. Yang J assisted in designing and performing the lipid

and content mixing experiments. Liu Q and de Vries JW synthesized lipid-

DNA. Gruszka A performed cryo TEM experiments. Rodríguez-Pulido A and

Crielaard BJ interpreted the data and prepared the manuscript. Kros A and

Herrmann A supervised the project. All authors edited the manuscript.


69

References

1. Rodríguez-Pulido, A.; Ortega, F.; Llorca, O.; Aicart, E.; Junquera, E.; A Physicochemical Characterization of the Interaction between DC-Chol/DOPE Cationic Liposomes and DNA. J. Phys. Chem. B 2008, 112, 12555-12565.

2. Liu, J.; Jiang, X.; Ashley, C.; Brinker, C. J.; Electrostatically Mediated Liposome Fusion and Lipid Exchange with a Nanoparticle-Supported Bilayer for Control of Surface Charge, Drug Containment, and Delivery. J. Am. Chem. Soc. 2009, 131, 7567-7569.

3. Peer, D.; Park, E. J.; Morishita, Y.; Carman, C. V.; Shimaoka, M.; Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target. Science 2008, 319, 627-630.

4. Chiu, H. C.; Lin, Y. W.; Huang, Y. F.; Chuang, C. K.; Chern, C. S.; Polymer Vesicles Containing Small Vesicles within Interior Aqueous Compartments and pH-Responsive Transmembrane Channels. Angew. Chem. Int. Ed. 2008, 47, 1875-1878.

5. Volodkin, D. V.; Skirtach, A. G.; Möhwald, H.; Near-IR Remote Release from Assemblies of Liposomes and Nanoparticles. Angew. Chem. Int. Ed. 2009, 48, 1807-1809.

6. Dudia, A.; Koҫer, A.; Subramaniam, V.; Kanger, J. S.; Biofunctionalized Lipid-Polymer Hybrid Nanocontainers with Controlled Permeability. Nano Lett. 2008, 8, 1105-1110.

7. Cisse, I.; Okumus, B.; Joo, C.; Ha, T.; Fueling protein–DNA interactions inside porous nanocontainers. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12646-12650.

8. Birkner J. P.; Poolman B.; Koҫer A (2012) Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 12944-12949.

9. Louhivuori, M.; Risselada, H. J.; Giessen, van der E.; Marrink, S. J.; Release of content through mechano-sensitive gates in pressurized liposomes. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 19856-19860.

10. Ma, M.; Bong, D.; Controlled Fusion of Synthetic Lipid Membrane Vesicles. Acc Chem Res. 2013, 46, 2988-2997.


12. Kong, L.; Askes, S. H. C.; Bonnet, S.; Kros, A.; Campbell, F.; Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes. Angew. Chem. Int. Ed. 2016, 55, 1396-1400.

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13. Chen. Y.; Sen, J.; Bathula, S. R.; Yang, Q.; Fittipaldi, R.; Huang, L.; Novel Cationic Lipid That Delivers siRNA and Enhances Therapeutic Effect in Lung Cancer Cells. Mol pharm. 2009, 6, 696-705.

14. Dutta, D.; Pulsipher, A.; Luo, W.; Yousaf, M. N.; Synthetic Chemoselective Rewiring of Cell Surfaces: Generation of Three-Dimensional Tissue Structures. J. Am. Chem. Soc. 2011, 133, 8704-8713.

15. Chen, H.; Kim, S.; Li L.; Wang, S.; Park, K.; Cheng, J,. Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6596-6601.

16. Mora, N. L.; Bahreman, A.; Valkenier, H.; Li, H.; Sharp, T. H.; Sheppard, D. N.; Davis, A. P.; Kros, A.; Targeted anion transporter delivery by coiled-coil driven membrane fusion. Chem. Sci. 2016, 7, 1768-1772.

17. Li, S.; Huang, L.; In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes. Gene Therapy 1997, 4, 891-900.

18. Torchilin, V. P.; Levchenko, T. S.; Rammohan, R.; Volodina, N.; Papahadjopoulos-Sternberg B.; D’Souza G. G. M.; Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome–DNA complexes. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1972-1977.

19. Dzau, VJ.; Mann, M. J.; Morishta, R.; Kaneda, Y.; Fusigenic viral liposome for gene therapy in cardiovascular diseases. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11421-11425.

20. Shi, N.; Pardridge, W. M.; Noninvasive gene targeting to the brain. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7567-7572.

21. Chen, Y. A.; Scheller, R. H.; SNARE-MEDIATED MEMBRANE FUSION. Nat. Rev. Mol. Cell Biol. 2001, 2, 98-106.

22. Brunger, A. T.; Structure and functionof SNARE and SNARE-interacting proteins. Quarterly Reviews of Biophysics 2006, 38, 1-47.

23. Stengel, G.; Zahn, R.; Höök, F.; DNA-Induced Programmable Fusion of Phospholipid Vesicles. J. Am. Chem. Soc. 2007, 129, 9584-9585.

24. Chan, Y. H. M.; Lengerich, van B.; Boxer, S. G.; Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 2008, 3, 17-21.

25. Chan, Y. H. M.; Lengerich, van B.; Boxer, S. G.; Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 979-984.

26. Lengerich, B.; Rawle, R. J.; Bendix, P. M.; Boxer, S. G.; Individual Vesicle Fusion Events Mediated by Lipid-Anchored DNA. Biophys J 2013, 105, 409-419.

27. Xu, W.; Wang, J.; Rothman, J. E.; Pincet, F.; Accelerating SNARE-Mediated Membrane Fusion by DNA–Lipid Tethers. Angew. Chem. Int. Ed. 2015, 54, 14388-14392.


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28. Stengel, G.; Simonsson, L.; Campbell, R. A.; Höök, F.; Determinants for Membrane Fusion Induced by Cholesterol-Modified DNA Zippers. J. Phys. Chem.B 2008, 112, 8264–8274.

29. Anaya, M.; Kwak, M.; Musser, A, J.; Müllen, K.; Herrmann, A.; Tunable Hydrophobicity in DNA Micelles: Design, Synthesis, and Characterization of a New Family of DNA Amphiphiles. Chem. Eur. J. 2010, 16, 12852-12859.

30. Marsden, H. R.; Tomatsu, I.; Kros, A.; Model systems for membrane fusion. Chem. Soc. Rev. 2011, 40, 1572-1585.

31. Versluis, F.; Voskuhl, J.; Kolck, van B.; Bremmer, M.; Albregtse, T.; Kros, A.; In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion. J. Am. Chem. Soc. 2013, 135, 8057-8062.

32. Alemdaroglu, F. E.; Ding, K.; Berger, R.; Herrmann, A.; DNA-Templated Synthesis in Three Dimensions: Introducing a Micellar Scaffold for Organic Reactions. Angew. Chem. Int. Ed. 2006, 45, 4206-4210.

33. Pfeiffer, I.; Höök, F.; Bivalent Cholesterol-Based Coupling of Oligonucletides to Lipid Membrane Assemblies. J. Am. Chem. Soc. 2004, 126, 10224-10225.

Chapter 4

DNA Replacement and Hybridization Chain

Reaction on the Surface of Liposome Membrane

This chapter has been submitted for publication.

Chapter 4

74

4.1 Introduction

Synthetic biology and cell surface engineering techniques in-vitro and in-

vivo have resulted in novel tools for the development of membrane

biology,1 offering promising membrane-based devices that may enable new

types of artificial tissues,2,3 biosensors,4,5 drug delivery approaches,6,7 3D

bio-printing and the study of lipid metabolism.8 To boost the development

of these technologies, there is a growing need to enhance surface

engineering techniques of membranes under in-vitro and in-vivo conditions

with particular emphasis on exploiting artificial surface receptors9 and

DNA Replacement and Hybridization Chain Reaction on the Surface of Liposome Membrane

75

designing novel biomaterials guided by natural processes,10 such as self-

assembling peptides, proteins and DNA oligonucleotides. Especially the

latter class of biomacromolecules is very appealing to fabricate complex

architectures because the sequence specific base pairing of

oligonucleotides allows the prediction of the resulting structure based on

the sequence composition, qualifying nucleic acids as indispensible

building blocks in soft matter nanotechnology.11 In conjunction with

advances in solid phase DNA synthesis methods,12 a plethora of

programmed 2- and 3-dimensional self-assembled architectures was

achieved.13,14

The facile chemical modification of oligonucleotides with hydrophobic

anchors also allowed the fabrication of DNA-based functional

membranes.15 In the context of liposomes, DNA hybridization-induced

vesicle aggregation,16 and fusion were realized.17 Photoresponsive DNA-

lipid assemblies, fabricated by either anchoring DNA with a azobenzene

moiety18 or hybridization of a photosensitizer mediated the cargo release

from liposomes.19 While these functions relied on simple DNA amphiphiles

that were inserted in the membrane, vesicle deformation and even

destruction of these containers was achieved with immobilizing and

polymerizing more complex DNA origami structures.13,20 Further extension

of these concepts led to a DNA-based atomistically determined molecular

valve capable of controlling transport of small molecules across a biological

membrane.21

In this chapter, we implement such membrane engineering related DNA

nanotechnology on the surface of a phospholipid bilayer. We performed

studies to establish the anchoring of DNA amphiphiles in such a bilayer,

subsequent hybridization, strand replacement and DNA hybridization

chain reaction (HCR). For that purpose, vesicles served as a model system.

Previously, hybridization of DNA on a liposome surface was

demonstrated,22 however, strand replacement and DNA HCR, to the best of

our knowledge, has never been performed in liposomal systems.

Chapter 4

76


4.2.1 Design and definition of lipid-DNA

For surface anchoring of oligonucleotides, we employed lipid-modified

DNA,23 consisting of a hydrophobic alkyl chain and an ethyne function

attached to the nucleobase, i.e. at the 5-position of uracil (Scheme 4.1A).

The incorporation of the hydrophobic building blocks was achieved as

phosphoramidites by solid phase synthesis employing an automated DNA

synthesizer and a previously established procedure.24 Due to this

convenient incorporation method, multiple hydrophobic nucleotides can be

introduced into the same oligonucleotide at any desired position allowing

to tune the interaction with phospholipid membranes. Here, we chose four

lipid-modified deoxyuridine units attached either to the 3’- or to 5’-end of

the oligonucleotide sequences, which are comprised of 18 or 28

nucleotides (Scheme 4.1B-D).

Scheme 4.1 Schematic representation of structures: (A) Chemical structure of lipid-modified

deoxyuridine (dU). (B) U4T-18, (C) U4T-28 and (D) CrU4T-18.


77

They are abbreviated as UxTy, where x represents the number of lipid-

modified uracils at the terminus while y denotes the overall number of

nucleotides of the sequence (Table 4.1). The four consecutive hydrophobic

anchoring units guarantee stable incorporation into a phospholipid

membrane of vesicles for at least 24 h as proven by a fluorometric assay,

which was described in Chapter 2. 24

Table 4.1 Sequences and modifications of DNA.

DNA Sequence ( 5’→3’) *

U4T-18 5’-UUUUGCGGATTCGTCTGC-3’

CrU4T-18 5’-GCAGACGAATCCGCUUUU-3’

U4T-28 5’-UUUUAATTGGGTGCGGCTTAGGATCTGA-3’

C488 5’-GCAGACAGGTCCGC-3’-ATTO488

C594 5’-GCAGACAGGTCCGCGTTTGT-3’-ATTO594

20-mer 5’-ACAAACGCGGATTCGTCTGC-3’

M1 5’-GTGCGGCTTAGGATCTGATGAAA

CTCAGATCCTAAGCCGCACCCAATT-3’

M1-FAM 6-FAM-5’-GTGCGGCTTAGGATCTGATGAAACTCA

GATCCTAAGCCGCACCCAATT-3’

M2 5’-GTTTCATCAGATCCTAAGCCGCACAAT

TGGGTGCGGCTTAGGATCTGA-3’

M2-Cy3 Cy3-5’-GTTTCATCAGATCCTAAGCCGCACAAT

TGGGTGCGGCTTAGGATCTGA-3’

*: U represents the modified uracil base. ATTO594, ATTO488, 6-FAM, Cy3 represent fluorescent dyes covalently bound to the DNA oligonucleotides.

4.2.2 DNA hybridization and replacement on the surface of liposomes

Firstly, U4T-18 was stably anchored into the membrane of vesicles

(diameter 120 nm) by in situ modification, i.e. by addition of a U4T-18

solution to plain liposomes (DOPC:DOPE:Cholesterol, 2:1:1, molar ratio).

Followed by 1h incubation at 50 °C. In situ modification spontaneously

Chapter 4

78

occurred due to the hydrophobic units part of lipid-DNA piercing into the

interior of the phospholipid bilayer. Then two Fluorescence Resonance

Energy Transfer (FRET) systems, Rhodamine Rh-DHPE/C594 and

C488/Rh-DHPE, were used to demonstrate the availability of the anchor

sequence for hybridization and DNA replacement (Fig 4.2A).

Fig. 4.2 (A) Schematic representation of DNA replacement on the surface of liposomes. (B)

Fluorescence spectra (λEX = 470 nm) of FRET system with U4T-18/Rh-DHPE/C594. FRET is

achieved when C594 hybridizes with U4T-18 to bring the donor Rh closer to the acceptor C594

(dashed purple line). Afterwards, C594 was peeled off from U4T-18 by hybridizing with 20-mer

(black line). Disruption of liposomes by addition of Triton X-100 (0.3% (v/v)) results in

termination of FRET (dashed red line). (C) After C594 was removed (black line Fig. 4.2B), U4T-

18 remained on the liposome and maintained the ability to hybridize with C488, which leads to

FRET between donor C488, and acceptor Rh-DHPE (black line). Liposome disassembly after the

addition of 0.3% (v/v) Triton X-100 results in an increase of C488 donor emission (dashed red

line).

In the Rh-DHPE/C594 system, ATTO594 was covalently attached to the 3’

end of a 20mer DNA (C594) to act as an acceptor. In parallel, rhodamine-

functionalized phospholipid (Rh-DHPE) was incorporated into the

liposomal bilayer to function as a donor (Fig 4.2A, step 1). As

demonstrated by a clear emission peak at 624 nm (C594, acceptor

fluorescence peak), hybridization only occurred upon mixing of C594 with


79

U4T-18-grafted Rh-DHPE-containing vesicles, positioning both dyes

sufficiently close to each other to achieve FRET (Fig 4.2B, dashed purple

line). Afterwards, a 20-mer DNA oligonucleotide that is fully

complementary to C594 was introduced to peel off C594 from U4T-18 (Fig

4.2A, step 2), which resulted in a higher signal of the donor emission of Rh-

DHPE (592 nm) and lowering of the acceptor emission (C594, 624 nm)

signal (Fig 4.2B, black line). Then, a 14-mer DNA which is complementary

to U4T-18 was covalently attached to ATTO488 and hybridized with free

U4T-18 (Fig 4.2A, step 3). In this case, C488 acted as donor for Rh-DHPE

which forms a second FRET system: C488/Rh-DHPE. Disruption of

liposomes by addition of Triton X-100 resulted in an increased donor peak

(C488, 520 nm) and slightly decreased acceptor peak (Rh-DHPE, 592 nm)

(Fig 4.2C), confirming that replacement by hybridization on the surface of

the vesicles was successfully achieved.

Fig. 4.3 Absence of FRET when liposomes are decorated with CrU4T-18. (A) Fluorescence

spectra (λEX = 470 nm) of non-FRET system with CrU4T-18/Rh-DHPE/C594 (dashed purple

line). After 1h incubation, 20-mer was added to the system (black line). Disruption of liposomes

by addition of Triton X-100 (0.3% (v/v)) results in termination of FRET (dashed red line). (B)

Fluorescence spectra of non-FRET system with CrU4T-18/C488/Rh-DHPE before (black line)

and after (dashed red line) addition of Triton X-100.

Chapter 4

80

In the control experiments involving CrU4T-18-liposomes, which cannot

hybridize with C594 or C488, similar emission spectra were obtained

before and after liposomal disruption, indicating that no FRET occurred in

the absence of complementary anchoring units on the vesicle surface (Fig.

4.3).

Moreover, native polyacrylamide gel electrophoresis (PAGE) was

performed to demonstrate DNA replacement in buffer in the absence of

liposomes (Fig. 4.4). The results showed that both C488 and C594

hybridize with U4T-18 (lane 4 and lane 5, respectively) and that the 20-

mer can efficiently peel off C594 from U4T-18 (lane 6). After C594 was

removed from U4T-18 by 20-mer, C488 hybridized with free U4T-18 (lane

7).

Fig. 4.4 PAGE of DNA replacement in buffer (M is maker). The sample run from left to right,

Lane 1: C488; Lane 2: C594; Lane 3: 20-mer; Lane 4: U4T-18 + C488; Lane 5: U4T-18 + C594;

Lane 6: C594 was peeled off from U4T-18 by 20-mer; Lane 7: C488 hybridized with free U4T-18

from Lane 6; Lane 8: C488 + 20-mer; Lane 9: C594 + 20-mer. The excitation with UV was at

366 nm.

4.2.2 DNA hybridization chain reaction (HCR) on the surface of

liposomes

Since initiation of HCR from a lipid membrane was not demonstrated

before we first established a HCR protocol for decorating the rim of

liposomes with a DNA layer. Membrane anchor U4T-28, a 28-mer lipid-


81

DNA with 4 modified lipid bases, was introduced to liposomes. Next,

hairpin strands M1 (partially complementary to U4T-28) and M2 (partially

complementary to M1) were added. Hybridization of M1 to U4T-28 results

in liberation of its loop that subsequently can hybridize with M2.25 Opening

of the M2 hairpin exposes a sequence that binds to a new M1 monomer

from the solution. In turn, opening of the M1 hairpin exposes a sequence

that can bind new M2. This effectively triggers the “supramolecular

polymerization” of M1 and M2 with surface anchor U4T-28 as initiator (Fig.

4.5).

Fig. 4.5 Schematic representation of lipid-DNA initiated HCR . a’, b’, and c’ are regions which

are complementary to regions a, b, and c, respectively. Hairpin M1 can be unfolded by

hybridization with initiator U4T-28 or open M2, while hairpin M2 can be unfolded by

hybridization with open M1, resulting in growing DNA strands.

Agarose gel electrophoresis analysis was used to prove U4T-28 initiated

HCR with M1 and M2 (Fig. 4.6). The hairpin sequences M1 and M2 (Fig. 4.6,

lane 1 and 2, respectively) did not hybridize in the absence of U4T-28 (Fig.

Chapter 4

82

4.6, lane 3). The chain length of the resulting duplex DNA is inversely

related to the initiator concentration (Fig. 4.6, lane 6-8).

Fig. 4.6 Agarose gel electrophoresis analysis of DNA HCR. Lane 1: M1; lane 2: M2; lane 3:

M1+M2; lane 4: M1+U4T-28; lane 5: M2+U4T-28; lanes 6–8: three different molar ratios of

initiator (1:1:1, 1:1:0.5, 1:1:2, M1:M2:U4T-28).

To prove DNA extension on the surface of liposomes, M2 was labeled with a

fluorophore (Cy3). Firstly, U4T-28 was incubated with liposomes for 30min

at 50 °C. PTHK polysulfone membrane filters (100 kDa) were used to

remove unincorporated U4T-28 by centrifugation. M1 and M2-Cy3 (two

equivalents in relation to U4T-28), were added to the system at room

temperature for 1 h, after which again a 100 kDa molecular weight cut off

filter was used in a centrifugation step to remove free M1 and M2-Cy3.

Afterwards, fluorescence intensity of the supernatant containing liposomes

was measured. The fluorescence spectra were recorded in the range of

530-620 nm with excitation at 520 nm for M2-Cy3 detection. A clear

emission peak at 566 nm (Cy3) only occurs upon chain reaction on the

surface of the liposomes (Fig. 4.7B, dashed red line), whereas in the

absence of M1 (Fig. 4.7B, dashed blue line) or U4T-28 (Fig. 4.7B, black

line), negligible Cy3 signals were observed.


83

Fig. 4.7 DNA HCR on the surface of liposomes. (A) Schematic representation of HCR for

decorating the outer layer of liposomes with a DNA shell. Liposomes (DOPC/DOPE/Chol = 2:1:1

mol%) decorated with U4T-28, and incubated with M1 and M2-Cy3 for 1 h, lead to DNA HCR

(I). After centrifugation and filtration, the fluorescence of supernatant containing liposomes

was measured. (B) Fluorescence spectra of Cy3 in HCR system (I, dashed red line). In the

absence of M1 (II, dashed blue line) or U4T-28 (III, black line), no fluorescence was observed.

4.3 Conclusion

In this chapter, we invented new concepts for the DNA functionalization of

a liposome surfaces. We explored DNA hybridization and the dynamic

exchange of DNA sequences on the surface of liposomes with two FRET

systems. As an anchoring unit a DNA amphiphile was utilized. Hydrophobic

units were incorporated into nucleobases, which pierce into the interior of

the phospholipid bilayer. DNA hybridization on the surface of liposomes

was proved by FRET between C594 (acceptor) and U4T-18-grafted Rh-

DHPE-containing (donor) vesicles, positioning both dyes sufficiently close

to each other to achieve FRET. After that, a 20-mer DNA oligonucleotide

that is fully complementary to C594 was introduced to peel off C594 from

U4T-18, which was confirmed by the increase of donor signal. Then, C488

hybridized with free U4T-18 acted as a donor for Rh-DHPE. The results

demonstrated that the hybridization process can be designed to be

Chapter 4

84

reversible allowing exchange of surface functionalities by simple addition

of DNA sequences. Finally, a DNA based amplification process was

performed atop of the liposome enabling the multiplication of surface

functionalities from a single DNA anchoring unit. A DNA probe, M2-Cy3,

was employed to detect the DNA HCR on the liposome’s surface. Compared

with control experiments, which were lacking DNA oligonucleotide

monomer for HCR or of the initiator, a significant stronger fluorescence

intensity of Cy3 was observed which can only be rationalized when

multiplication of DNA occurs on surface of liposomes. The hybridization

chain reaction preformed in this chapter allows accumulation of multiple

cargoes or signals on the liposomal surface by using anchored single DNA

strands. The experiments shown in this chapter significantly extend the

functionality of liposomal system regarding loading and decorating the

vesicle surface. This might be exploited in the fields of drug delivery or

diagnostics.


4.4.1 Materials



from Avanti Polar Lipids (Alabaster, USA) (purity >99 %) and used without

further purification. Headgroup-labeled phospholipid, lissamine rhodamine

B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammoni

-um salt) (Rh-DHPE) was purchased from Invitrogen (Amsterdam,

Netherlands), and used as received. PTHK polysulfone membrane filters

with a NMWL of 100 kDa were purchased from Sigma-Aldrich. The DNA-

dye conjugates C488, C594 and M2-Cy3 were purchased from Biomers.net

GmbH (Ulm, Germany). Triton X-100 (10% in water) was purchased from

Sigma-Aldrich (St. Louis, United States). Anhydrous CHCl3 was acquired

from Acros Organics (Geel, Belgium) and stored over molecular sieves. For

all experiments, ultrapure water (specific resistance > 18.4 MΩ cm) was

obtained by a Milli-Q water purification system (Sartorius).


85

4.4.2 Synthesis and characterization of amphiphilic oligonucleotides

Fig. 4.8 MALDI-TOF mass spectra of (A) U4T-18, (B) U4T-28 and (C) CrU4T-18.

Fig. 4.9 RPC HPLC analysis of purified lipid-DNAs: (A) U4T-18, (B) U4T-28 and (C) CrU4T-18.

Numbers beside the elution peaks represent the buffer B contents when lipid-DNAs were eluted.

Chapter 4

86

The amphiphilic oligonucleotides were synthesized and purified as

reported previously (see Chapter 2).23,26 The identity and purity of the

oligonucleotides were confirmed by RPC-HPLC and MALDI-TOF mass

spectrometry (Fig. 4.8 and Fig. 4.9).


Firstly, chloroform was removed from lipid mixture (DOPC:DOPE:

Cholesterol, 2:1:1, molar ratio) by evaporation under an air stream and

then under vacuum overnight. The dried lipid mixture was dissolved in an

aqueous PBS buffer (150 mM NaCl, 15 mM K2HPO4, 5 mM KH2PO4) by 5

cycles of vortexing and freeze-thawing. Subsequently, the sample was

dispersed by extruding 21 times using an extruder and 100 nm

polycarbonate membranes (Whatman) to obtain large unilamellar vesicles

(LUVs), after which the liposomes with lipid-DNA were incubated at 50 °C

for 1h. All lipid-DNA liposomes were used within one day, and had an

average diameter of around 120 nm as determined by DLS (ALV/CGS-3

ALV-Laser Vertriebsgesellschaft mbH, Langen, Germany). The molar ratio

between lipid and U4T-18 was 500:1.

4.4.3 DNA replacement on liposomes measured by Fluorescence

Resonance Energy Transfer (FRET) assay

U4T-18 was incorporated in Rh-DHPE/(DOPC+DOPE) (3:97 molar ratio)

liposomes to obtain U4T-18 liposomes with a lipid to U4T-18 ratio of 500:1.

Subsequently, an aliquot of these liposomes was mixed with a small

amount of Cr-ATTO488 or CrATTO594 such that [U4T-18] = [Cr-ATTO488]

= [Cr-ATTO594] = 0.906 μM and with a final lipid (DOPC+DOPE)

concentration of 0.45 mM. Fluorescence emission spectra of

donor/acceptor, CrATTO488/Rh-DHPE or Rh-DHPE/CrATTO594 in the

500–700 nm region, were recorded with excitation at 470 nm using a

SpectraMax M3 (Molecular Devices) fluorescence spectrophotometer.

Measurements were carried out at a constant temperature of 25 °C.


87

4.4.4 Native polyacrylamide gel electrophoresis to detect DNA

replacement

Native polyacrylamide gel electrophoresis was performed using a 15% gel

made with TBE buffer (90 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0)

and run at 120 V for 100 min.

4.4.5 Agarose gel electrophoresis to monitor HCR

The 2 % agarose gel was prepared by using SB buffer (10 mM NaOH, pH

adjusted to 8.5 with boric acid) 27 and run at 90 V for 3 h. After

electrophoresis, the agarose gel was stained with ethidium bromide and

visualized under UV light.

4.4.6 DNA hybridization chain reaction on the surface of liposomes

After incubation of 0.45 mM liposomes with 0.906 μM U4T-28 at 50 °C for

30 min, PTHK polysulfone membrane filters (100 kDa) were used to

remove unincorporated U4T-28 by centrifugation at 1000 rpm (rotor: FA-

45-18-11) for 30 min. M1 and M2-Cy3 (two equivalents in relation to U4T-

28) were added to the system at RT for 1 h, after which again a 100 kDa

molecular weight cut off filter was used in a centrifugation step to remove

free DNA (1000 rpm, 30 min). After re-suspension and centrifugation, the

supernatant was washed twice with PBS buffer. Afterwards, the

fluorescence spectra were recorded in the range of 530-620 nm with

excitation at 470 nm for M2-Cy3, using a SpectraMax M3 (Molecular

Devices) fluorescence spectrophotometer. Measurements were carried out

at 25 °C.


Meng Z designed, conducted the experiments and performed data analysis.

Meng Z and Yang J prepared the manuscript. Liu Q synthesized lipid-DNA.

Kros A and Herrmann A supervised the project.

Chapter 4

88

References

1. Saeui, C. T.; Mathew, M.P.; Liu, L.; Urias, E.; Yarema, K. J.; Cell Surface and Membrane Engineering: Emerging Technologies and Applications. J. Funct. Biomater. 2015, 6, 454-485.

2. Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M.; Taking cell matrix adhesions to the third dimension. Science 2001, 294, 1708-1712.

3. Revzin, A.; Rajagopalan, P.; Tilles, A. W.; Berthiaume, F.; Yarmush, M. L.; Toner, M.; Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography. Langmuir 2004, 20, 2999-3005.

4. Shear, J. B.; Fishman, H. A.; Allbritton, N. L.; Garigan, D.; Zare, R. N.; Scheller, R. H.; Single cells as biosensors for chemical separations. Science 1995, 267, 74-77.

5. Rider, T. H.; Petrovick, M. S.; Nargi1, F. E.; Harper, J. D.; Schwoebel, E. D.; Mathews1, R. H.; Blanchard, D. J.; Bortolin, L. T.; Young, A. M.; Chen, J.; Hollis, M. A.; A B cell-based sensor for rapid identification of pathogens. Science 2003, 301, 213-215.

6. Yang, J.; Shimada, Y.; Olsthoorn, R. C. L.; Snaar-Jagalska, B. E.; Spaink, H. P.; Kros, A.; Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model. ACS Nano. 2016, 10, 7428-7435.

7. Yang, J.; Bahreman, A.; Daudey, G.; Bussmann, J.; Olsthoorn, R. C. L.; Kros, A.;.; Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes. ACS Cent. Sci. 2016, 2, 621-630.

8. Han, G. S.; O'Hara, L.; Carman, G. M.; Siniossoglou, S.; An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth. J. Biol. Chem. 2008, 283, 20433-20442.

9. Mrksich, M.; What can surface chemistry do for cell biology? Curr. Opin. Chem. Biol. 2002, 6, 794-797.

10. Zhang, S.; Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 2003, 21, 1171-1178.

11. Seeman, N. C.; DNA in a material world. Nature 2003, 421, 427-431.

12. Caruthers, M. H.; Gene synthesis machines: DNA chemistry and its uses. Science 1985, 230, 281-285.

13. Kocabey, S.; Kocabey, S.; Kempter, S.; List, J.; Xing, Y.; Bae, W.; Schiffels, D.; Shih, W. M.; Simmel, F. C.; Liedl, T.; Membrane-Assisted Growth of DNA Origami Nanostructure Arrays. ACS Nano. 2015, 9, 3530-3539.


89

14. Todhunter, M.E.; Jee, N. Y.; Hughes, A. J.; Coyle, M. C.; Cerchiari, A.; Farlow, J.; Garbe, J. C.; LaBarge, M. A.; Desai, T. A.; Gartner, Z. J.; Programmed synthesis of three-dimensional tissues. Nat. Methods 2015, 12, 975-981.

15. Yoshina-Ishii, C.; Boxer, S.G.; Arrays of Mobile Tethered Vesicles on Supported Lipid Bilayers. J. Am. Chem. Soc. 2003,125, 3696-3697.

16. Jakobsen, U.; Simonsen, A.C.; Vogel, S.; DNA-Controlled Assembly of Soft Nanoparticles. J. Am. Chem. Soc. 2008, 130, 10462-10463.

17. Stengel, G.; Zahn, R.; Höök, F.; DNA-Induced Programmable Fusion of Phospholipid Vesicles. J. Am. Chem. Soc. 2007, 129, 9584-9585.

18. Hernández-Ainsa, S.; Ricci, M.; Hilton, L.; Aviñó, A.; Eritja, R.; Keyser, U. F.; Controlling the Reversible Assembly of Liposomes through a Multistimuli Responsive Anchored DNA. Nano Lett. 2016, 16, 4462-4466.


20. Czogalla, A.; Czogalla, A.; Kauert, D. J.; Franquelim, H. G.; Uzunova, V.; Zhang, Y.; Seidel, R.; Schwille, P.; Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane Vesicles. Angew. Chem. Int. Ed. 2015, 127, 6601-6605.

21. Burns, J. R.; Seifert, A.; Fertig, N.; Howorka, S.; A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane. Nat. Nanotechnol. 2016, 11, 152-156.

22. Kurz, A.; Bunge, A.; Windeck, A.; Rost, M.; Flasche, W.; Arbuzova, A.; Strohbach, D.; Müller, S.; Liebscher, J.; Huster, D.; Herrmann, A.; Lipid-Anchored Oligonucleotides for Stable Double-Helix Formation in Distinct Membrane Domains. Angew. Chem. Int. Ed. 2006, 45, 4440-4444.

23. Anaya, M.; Kwak, M.; Musser, A. J.; Mullen, K.; Herrmann, A.; Tunable hydrophobicity in DNA micelles: design, synthesis, and characterization of a new family of DNA amphiphiles. Chem. Eur. J. 2010, 16, 12852-12859.

24. Meng, Z.; Yang, J.; Liu, Q.; de Vries J. W.; Gruszka, A.; Rodríguez-Pulido, A.; Crielaard, B J.; Kros, A.; Herrmann, A.; Efficient Fusion of Liposomes by Nucleobase Quadruple-Anchored DNA. Chem. Eur. J. 2017, DOI: 10.1002/chem.201701379.

25. Dirks, R. M.; Pierce, N. A.; Triggered amplification by hybridization chain reaction. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15275-15278.

26. Kwak, M.; Minten, I. J.; Anaya, D. M.; Musser, A. J.; Brasch, M.; Nolte, R. J. M.; Müllen, K.; Cornelissen, J. J. L. M.; Herrmann, A.;Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers. J. Am. Chem. Soc. 2010, 132, 7834-7835.

27. Brody, J. R.; Kern, S. E.; Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. BioTechniques 2004, 36, 214-216.

Chapter 5

Performing DNA Nanotechnology Operations

on a Zebrafish Surface

This chapter has been submitted for publication.

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5.1 Introduction

Zebrafish is an ideal model vertebrate system with robust, external, and

transparent development allowing sophisticated imaging and experimental

techniques. In just a few days, embryos develop from single cells to

remarkably complex body structures and organ systems (Fig. 5.1).

Zebrafish embryos exhibit the great feature that they develop as "see

through" embryos, meaning that, all internal development can be clearly

observed from the outside in the living system.1

Fig. 5.1 Timeline of zebrafish embryonic development during 72 hours.

Fluorescent probes, such as synthetic organic dyes,2,3 fluorescent

proteins,4,5 and quantum dots,6-8 have been used extensively to monitor

biomolecules and biologically relevant species in vitro and in vivo. Due to

their transparent properties, zebrafish embryos have received great

attention for live vertebrate imaging due to the possibility to conduct high

resolution in vivo imaging. Compared to mammals, many structures and

processes are similar in zebrafish such as the brain and spinal cord.

Because of its outstanding suitability for imaging and transgenesis

approaches, the developing zebrafish has become a leading vertebrate

model for studies in drug discovery and a variety of human diseases.9,10 In

many cases, molecular imaging using fluorescent probes has been carried

out within cells. However, live cell imaging is not completely acceptable for

obtaining detailed information about the biological effects of analytes in a

tissue context. In contrast, images of the insides of live animals provide a

more informative view of these effects. Moreover, fluorescent imaging

Performing DNA Nanotechnology Operations on a Zebrafish Surface

93

studies using organisms that are genetically close to humans have become

highly attractive (Fig. 5.2).11

Fig. 5.2 Live cell and animal imaging using fluorescent probes. The trend for detection of

analytes has changed from live cell imaging to whole animal imaging.11

Owing to its small size, optical transparency, external fertilization and easy

manipulation, zebrafish are perhaps the most suitable vertebrate model

animals for in vivo imaging using fluorescent probes.

In this chapter, we implemented a membrane engineering related DNA

nanotechnology on the surface of a living animal. We investigated whether

it was possible to insert the lipid-modified DNA sequences into the

membrane of live zebrafish to function as an artificial receptor. Firstly, the

immobilization of membrane-anchor-functionalized oligonucleotides on a

zebrafish was demonstrated. Then, functionalization of protruding single-

stranded DNA atop the fish was realized by Watson-Crick base pairing

employing complementary DNA sequences. In this way, small molecules

and liposomes were guided and attached to the fish surface. The anchoring

process can be designed to be reversible allowing exchange of surface

functionalities by simple addition of DNA sequences. Finally, a DNA based

amplification process was performed atop of the zebrafish enabling the

multiplication of surface functionalities from a single DNA anchoring unit.

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5.2.1 DNA hybridization on a zebrafish surface and strand replacement

A DNA-based receptor can be used to modify the cell membrane with new

functions, such as for immobilization of surface probes or other payloads

for targeted delivery onto or through the lipid bilayer. For that purpose, 1

µM lipid-DNA U4T-18 was utilized and incubated with zebrafish embryos

of 1 day post-fertilization (dpf) for 1 h. Previously the group of Irvine12

performed cell membrane insertion of oligonucleotides carrying diacyl-

lipid (C18) or cholesterol anchors using an incubating time of 30 min.

Because electrostatic repulsion might delay incorporation into the

membrane, we prolonged the time for incubation of lipid-DNA with

zebrafish membrane to 1 h.

Subsequently, the 20mer oligonucleotide C594, which is partially

complementary to U4T-18 and contains the red emitting fluorophore

ATTO594, was added to hybridize to the lipid-modified surface anchor.

Fluorescence microscopy showed staining of the fish surface as evidenced

by the red emission (Fig. 5.3A) indicating that U4T-18 was successfully

incorporated into the fish skin and that the protruding single-stranded

DNA chain can selectively undergo sequence specific hybridization.

Next, we investigated if it is possible to dynamically exchange the red label

by a removal strand. This strategy of strand replacement was previously

introduced in the context of a DNA fueled molecular machine.13 Here, we

employed this strategy for the reversible and gentle labeling of the skin of a

living animal. Therefore, 2 µM 20-mer DNA oligonucleotide that is fully

complementary to C594 was introduced to peel off C594 from U4T-18. The

removal of C594 was proven by disappearance of red fluorescence on the

fish surface (Fig. 5.3B). After removal of C594, U4T-18 remained on the

skin and kept the ability to hybridize with other complementary DNA

sequence such as C488, a 14mer DNA oligonucleotide complementary to

U4T-18 and labeled with the green emitting fluorophore ATTO488. Clear

evidence for the strand replacement was the green fluorescence observed

on the exterior of the fish (Fig. 5.3C). Control experiments in which U4T-18


95

lipid-DNA or 20-mer oligonucleotides were omitted (Fig. 5.4) showed no

(changes in) fluorescence.

Fig. 5.3 Fluorescent labelling and DNA replacement on the surface of zebrafish embryos. (A)

Lipid DNA (U4T-18) is anchored on the skin membrane of 1 dpf zebrafish embryos and

hybridizes to 1 µM ATTO594 fluorescently labeled complementary DNA (C594), resulting in red

fluorescence on the zebrafish surface. (B) A 20-mer oligonucleotide replaces C594 by means of

strand displacement, resulting in loss of fluorescence of the fish. (C). Addition of 1 µM ATTO488

fluorescenlyt labeled complementary DNA (C488) hybridizes with U4T-18, results in

hybridization with U4T-18 and the appearance of a green fluorescence at the zebrafish surface.

Red channel = ATTO594; Green channel = ATTO488.

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Fig. 5.4 Fluorescence images of two control experiments: (A) untreated 2dpf zebrafish without

DNA anchoring unit was incubated with 1 µM ATTO594 labeled complementary DNA (C594)

for 1h. No fluorescence labelling was detected. (B) 2dpf zebrafish was decorated with U4T-18,

incubated with 1 µM C594 for 1h, washed three times with egg water, and subsequently

exposed to solution containing 1 µM ATTO488 labeled complementary DNA (C488). Although

the green labelled DNA was added to the fish, red fluorescence was detected on its surface. This

is due to lack of the removal strand. After all treatments of the fish with DNA, washing three

times with egg water was performed. Red channel = ATTO594; Green channel = ATTO488.

5.2.2 Loading larger containers to the zebrafish surface by Watson-

Crick base pairing

After demonstrating that base pairing is a very efficient tool to attach small

oligonucleotides to the live animal surface, we attempted to load larger

cargo to the zebrafish membrane. Therefore, phospholipid bilayer of

liposomes of 120 nm diameter was loaded with rhodamine-functionalized

phospholipid (Rh-DHPE), which is characterized by a red emission.

Likewise, the surface of the vesicles was decorated with lipid-modified

DNA that is complementary to that on the zebrafish (Fig. 5.5A). Proof of

successful loading of the fish surface by supramolecular bonds was

provided by fluorescence microcopy showing characteristic red

fluorescence originating from the liposomes, which are bound to the fish

surface (Fig. 5.5B). This result opens the way for potential DNA-mediated

delivery of liposomal cargo. These experiments demonstrate that the DNA

hybridization overcomes the repulsive hydration forces between the lipid


97

head groups and brings the two lipid bilayers with complementary DNA in

close proximity to achieve surface docking (aggregation).

Fig. 5.5 DNA duplex formation between U4T-18 and CrU4T-18 decorated liposomes on the

surface of zebrafish. (A) Schematic representation of liposomes docking on the surface of

zebrafish embryos by lipid-DNA hybridization. Confocal images of 2dpf zebrafish treated with

(B) U4T-18 for 1 h, followed by incubation with CrU4T-18 decorated liposomes or (C) treated

with 1 µM CrU4T-18 decorated liposomes in absence of U4T-18. The concentration of total

lipids (DOPC:DOPE:CHO= 2:1:1mol%) was 0.5 mM. Red Channel: Rh-DHPE.

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5.2.3 Nucleic acid mediated amplification process on live fish surface

To demonstrate the broad versatility of zebrafish surface engineering

enabled by lipid-DNA, we performed a DNA-based amplification process on

the animal, i.e. hybridization chain reaction (HCR). Previously, this method

was employed for augmenting the signal during nucleic acid detection.14

Later, this technique was utilized for surface modification with DNA

hydrogels.15 Here, we demonstrate that this supramolecular

polymerization can be performed on the exterior of the living animal.

Since initiation of HCR from a lipid membrane was not demonstrated

before we first established a HCR protocol for decorating the rim of

liposomes with a DNA layer (see chapter 4). Then, the optimized DNA

anchors and sequences were employed for modification of the fish surface

(Fig. 5.6). Compared to the previous experiments, for membrane anchoring

U4T-28, a 28mer lipid-DNA with 4 modified lipid bases, was bound to

zebrafish skin. Next, hairpin strands M1 (partially complementary to U4T-

28) and M2 (partially complementary to M1) were added (sequences see

chapter 4).16 Hybridization of M1 to U4T-28 results in liberation of its loop

that subsequently can hybridize with M2. Opening of the M2 hairpin

exposes a sequence that binds to a new M1 monomer from the solution. In

turn, opening of the M1 hairpin exposes a sequence that can bind new M2.

This effectively triggers the “supramolecular polymerization” of M1 and M2

with surface anchor U4T-28 as initiator, leading to extended DNA on the

zebrafish membrane (Fig. 5.6A). The realization of HCR was investigated

on the membrane of 1 dpf zebrafish embryo. As shown schematically in Fig.

5.6B, U4T-28 at the concentration of 1 µM was exposed to zebrafish

embryos for 1 h, followed by the incubation with a mixture of 2 µM M1-

FAM and 2 µM M2-Cy3 for 2 h. Both monomers were labeled with two

different fluorophores (FAM and Cy3). Green and red fluorescence could be

clearly observed due to progression of polymerization of M1-FAM and M2-

Cy3 from the initiator (Fig. 5.6C).


99

Fig. 5.6 DNA hybridization chain reaction (HCR) on the surface of zebrafish embryos. (A)

Schematic representation of DNA HCR on the surface of zebrafish embryos. a’, b’, and c’ are

regions that are complementary to regions a, b, and c, respectively. Hairpin M1 can be

unfolded by hybridization with initiator U4T-28, resulting in growing DNA strands. (B)

Addition of M1-FAM and M2-Cy3 to the U4T-28 pre-treated zebrafish resulted in DNA HCR and

concomitant increase in fluorescence. (C) Fluorescence images of 1 dpf zebrafish embryos after

incubation with U4T-28 for 1 h, and subsequent exposure to 1 µM M1-FAM and M2-Cy3 for 1 h.

Green channel: 6-FAM; Red channel: Cy3. (D) Normalized fluorescence intensity of attached

DNA on the surface of zebrafish embryos. Fluorescence intensities of images (C) and Fig.5.7A

were calculated by Image J and plotted as a percentage relative to the fluorescence of M1-FAM

or M2-Cy3 of Fig. 5.6C. The intensities of Fig. 5.6C were set to 100%.

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100

Two control experiments were performed to prove the HCR was initiated

on the membrane of zebrafish (Fig. 5.7). As shown in Fig. 5.7A, free M1-

FAM was washed away before the addition of M2-Cy3. In this case, the

fluorescent signals of M1-FAM and M2-Cy3 were 10 and 20 times lower,

respectively, than those obtained in the presence of HCR (Fig. 5.6D). Also,

when the monomer M1-FAM was omitted the HCR could not proceed and

consequently no fluorescence could be detected on the fish surface (Fig

5.7B).

Fig. 5.7 HCR on zebrafish skin depends on DNA hybridization between U4T-28, M1 and M2.

Fluorescence images of 1 dpf zebrafish (A) that were first incubated with U4T-28 and M1-FAM

for 2 hours, subsequently washed 3 times with egg water before the addition of M2-Cy3, or (B)

similar to (A) but without M1-FAM. Green channel: 6-FAM; Red channel: Cy3.

The signal increase by HCR was also clearly demonstrated by an

experiment involving non-fluorescent M2 and M1-FAM. In case of HCR

approximately 10-fold stronger green fluorescence was observed on the

surface of 2 dpf zebrafish embryos (Fig. 5.8A) compared to labeling with a

single fluorophore per anchor unit (Fig. 5.8B).


101

Fig. 5.8 In vivo DNA HCR enhances the fluorescence intensity of labeling. (A) Fluorescence

images of 2dpf zebrafish embryos that were first decorated with U4T-28, followed by 3 times

washing with egg water, incubation with M1-FAM for 1 h and M2 for another 1 h. (B) Only

FAM fluorescent labeled M1 (M1-FAM) was added after anchoring of U4T28 on the fish and

washing three times with egg water. Green channel: M1-FAM.

5.3 Conclusion

Previously, oligonucleotides were covalently attached to live cells by

metabolic oligosaccharide engineering allowing the introduction of

orthogonal chemical handles on the cell surface for DNA anchoring without

dependence on endogenous receptors.17 Besides oligosaccharides, cell-

surface proteins were exploited for the chemical modification of cells with

DNA.18,19 Similarly, cell-surface proteins were decorated with DNA by non-

covalent interactions.20 An alternative strategy for introducing artificial

DNA receptors on live cell surfaces represents the utilization of

oligonucleotides carrying hydrophobic membrane anchors, as described in

this study.21 Based on such an anchoring strategy, Watson-Crick base

pairing was exploited for the programmed synthesis of three-dimensional

tissues.22 The examples above demonstrate that anchored DNA in a lipid

bilayer developed into a powerful tool for realizing exciting functionalities

in the context of synthetic and natural membranes, even including live cells.

On the other hand, DNA nanostructures were employed in higher

organisms in the context of functional in-vivo imaging23 and for the

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targeted delivery of siRNA.24 To the best of our knowledge, there is no

experiment about DNA-based membrane engineering in a living animal

involving a wide variety of functions yet.

In this chapter, we demonstrated that lipid-DNA sequences with four

anchoring units could be readily incorporated in the surface layer of

zebrafish embryos. The single-stranded DNA present on the surface can be

functionalized by Watson-Crick base pairing enabling the sequence specific

functionalization of the live animal with small molecules or larger cargos,

for example, liposomal systems. The payloads connected by the

supramolecular tether DNA can be reversibly removed employing a

removal strand, which represents a very mild stimulus just requiring the

addition of a DNA sequence not affecting the life of the fish. Finally, we

successfully demonstrated the performance of a DNA mediated

amplification process on the fish skin. The hybridization chain reaction

allows attachment of multiple moieties on a single anchored DNA strand

allowing multiplication of cargoes or signals on the surface. Moreover, it

was shown that surface modification of model membranes in form of

liposomes by various DNA nanotechnology procedures could be easily

transferred to the live animal. This allows establishment of DNA based

surface functionalization procedures and their facile and fast

implementation in zebrafish. However, challenges in the application of

lipid-DNA in the transition of zebrafish to higher mammals remain. For

instance, the DNA part in lipid-DNA is susceptible to the deoxyribonuclease

in the circulation system of mammals. On the other hand, unlike zebrafish,

mammals are not clear and transparent, which makes the direct

visualization of fluorescently labeled tissues impossible. Nevertheless, due

to the broad application of zebrafish as animal model in drug development,

toxicology and nanoparticles characterization,25 we believe that the

platform presented here allows amalgamation of DNA nanotechnology

tools with live animals and enables efficient bio-barcoding as well as in vivo

tracking.


103

5.4 Experiment Section

5.4.1 Materials



from Avanti Polar Lipids (Alabaster, USA) (purity >99%) and used without

further purification. Headgroup-labeled phospholipid, lissamine rhodamine

B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammoni

-um salt) (Rh-DHPE) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-

dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium

salt) (NBD-DHPE) were purchased from Invitrogen (Amsterdam,

Netherlands), and used as received. The DNA-dye conjugates C488, M1-

FAM and M2-Cy3 were purchased from Biomers.net GmbH (Ulm, Germany).

Anhydrous CHCl3 was purchased from Acros Organics (Geel, Belgium) and

stored over molecular sieves.

5.4.2 Zebrafish strain, husbandry, and egg collection

Wildtype zebrafish were used in this study, and were maintained and

handled according to the guidelines from http://zfin.org. Fertilization was

performed by natural spawning at the beginning of the light period, and

eggs were raised at 28 °C. All experimental procedures were conducted in

compliance with the directives of the animal welfare committee of the

Leiden University.

5.4.3 Microscopy images

Zebrafishes were seeded in a glass bottom flask with egg water. After

incubation for 1 h with lipid-DNA, they were washed three times and then

incubated with other DNA oligonucleotides for 1 h. For live imaging,

zebrafish embryos were anaesthetized with 0.003% tricaine and mounted

on 0.6% low-melting agarose. Fluorescent images were acquired using a

Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems,

Wetzlar, Germany) and merged with Leica application suite advanced

fluorescence software (Leica Microsystems) or ImageJ software (National

Institutes of Health, Bethesda, MD, USA). A Leica MZ16FA stereo

microscope was used for stereo images. Images were adjusted for

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104

brightness and contrast using ImageJ. The wavelength settings for C488

were Ex/Em: 495/520nm (Ex laser: 488 nm), for C594 Ex/Em: 601/627

nm (Ex laser: 532 nm), for M1-FAM Ex/Em: 494/518 nm (Ex laser: 488

nm), and M2-Cy3 Ex/Em: 550/570 nm (Ex laser: 532 nm).


Yang J conducted the experiments and performed data analysis. Yang J and

Meng Z designed the experiments and prepared the manuscript. Liu Q

synthesized lipid-DNA. Yasuhito S produced the zebrafish embryo. Herman

S and René CLO interpreted the data. Kros A and Herrmann A supervised

the project. All authors edited the manuscript.


105

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19. Zhao, W.; Loh, W.; Droujinine, I. A.; Teo, W.; Kumar, N.; Schafer, S.; Cui, C. H.; Zhang, L.; Sarkar, D.; Karnik, R.; Karp, J. M.; Mimicking the inflammatory cell adhesion cascade by nucleic acid aptamer programmed cell-cell interactions. FASEB J 2011, 25, 3045-3056.

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21. Selden, N. S.; Todhunter, M. E.; Jee, N. Y.;Liu, J. S.; Broaders, K. E.; Gartner, Z. J.; Chemically Programmed Cell Adhesion with Membrane-Anchored Oligonucleotides. J. Am. Chem. Soc. 2012, 765-768.

22. Todhunter, M. E.; Jee, N. Y.; Hughes, A. J.; Coyle, M. C.; Cerchiari, A.; Farlow, J.; Garbe, J. C.; LaBarge, M. A.; Desai, T. A.; Gartner, Z. J.; Programmed synthesis of three-dimensional tissues. Nat Methods. 2015,12, 975-981.

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25 Evensen, L.; Johansen, P. L.; Koster, G.; Zhu, K.; Herfindal, L.; Speth, M.; Fenaroli, F.; Hildahl, J.; Bagherifam, S.; Tulotta, C.; Prasmickaite, L.; Mælandsmo, G. M.; Snaar-Jagalska, E.; Griffiths, G.; Zebrafish as a model system for characterization of nanoparticles against cancer. Nanoscale 2016, 8, 862-877.

http://pubs.acs.org/author/Trifonov%2C+Alexander

http://pubs.acs.org/author/Cecconello%2C+Alessandro

http://pubs.acs.org/author/Guo%2C+Weiwei

http://pubs.acs.org/author/Fan%2C+Chunhai

http://pubs.acs.org/author/Willner%2C+Itamar

http://pubs.acs.org/author/Shum%2C+Betty+J

http://pubs.acs.org/author/Onoe%2C+Hiroaki

http://pubs.acs.org/author/Douglas%2C+Erik+S

http://pubs.acs.org/author/Gartner%2C+Zev+J

http://pubs.acs.org/author/Mathies%2C+Richard+A

http://pubs.acs.org/author/Bertozzi%2C+Carolyn+R

http://pubs.acs.org/author/Francis%2C+Matthew+B

http://pubs.acs.org/author/Todhunter%2C+Michael+E

http://pubs.acs.org/author/Jee%2C+Noel+Y

http://pubs.acs.org/author/Liu%2C+Jennifer+S

http://pubs.acs.org/author/Broaders%2C+Kyle+E



https://www.ncbi.nlm.nih.gov/pubmed/?term=Jee%20NY%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Hughes%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Coyle%20MC%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Cerchiari%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Farlow%20J%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Garbe%20JC%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=LaBarge%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Desai%20TA%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

https://www.ncbi.nlm.nih.gov/pubmed/?term=Gartner%20ZJ%5BAuthor%5D&cauthor=true&cauthor_uid=26322836

http://www.nature.com/nnano/journal/v7/n6/abs/nnano.2012.73.html#auth-2

















http://pubs.rsc.org/-/results?searchtext=Author%3APatrick%20L.%20Johansen

http://pubs.rsc.org/-/results?searchtext=Author%3AGerbrand%20Koster

http://pubs.rsc.org/-/results?searchtext=Author%3AKaizheng%20Zhu

http://pubs.rsc.org/-/results?searchtext=Author%3ALars%20Herfindal

http://pubs.rsc.org/-/results?searchtext=Author%3AMartin%20Speth

http://pubs.rsc.org/-/results?searchtext=Author%3AFederico%20Fenaroli

http://pubs.rsc.org/-/results?searchtext=Author%3AJon%20Hildahl

http://pubs.rsc.org/-/results?searchtext=Author%3AShahla%20Bagherifam

http://pubs.rsc.org/-/results?searchtext=Author%3AClaudia%20Tulotta

http://pubs.rsc.org/-/results?searchtext=Author%3ALina%20Prasmickaite

http://pubs.rsc.org/-/results?searchtext=Author%3AGunhild%20M.%20M%C3%A6landsmo

http://pubs.rsc.org/-/results?searchtext=Author%3AEwa%20Snaar-Jagalska

http://pubs.rsc.org/-/results?searchtext=Author%3AGareth%20Griffiths

Summary

Summary

108

Summary

Great efforts have been dedicated to use DNA as a building block in

nanotechnology because of its versatile properties, such as high specificity

and programmability to form complex structures. The rapid development

of chemical methodology has been proven to be useful not only for the

preparation of pristine DNA and RNA, but also to make base, or backbone

modifications of the oligonucleotides as well as many analogues. The

structural variety has been further expanded from 2D-patterns or 3D-nano-

objects, which are basically fabricated with pristine DNA, to DNA hybrids,

such as DNA-nanoparticle hybrids including inorganic or soft materials.

Specially, the phosphoramidite chemistry has been adapted for the

modification of DNA with hydrophobic polymers or lipids, yielding DNA

amphiphiles. These DNA amphiphiles can be used to perform surface

modifications of biological membranes.

In Chapter 2, we have established a new strategy for anchoring

oligonucleotides in vesicle membranes enabled by attaching hydrophobic

units to the nucleobase. The membrane anchors are incorporated into the

oligonucleotide by automated solid phase synthesis allowing precise

control over the position and number of hydrophobic units within a DNA

sequence. A FRET system was used to prove the incorporation and stability

of lipid-DNA on the liposomal membrane. Single-stranded DNA

functionalized with four lipid-modified nucleobases was stably grafted

onto the membrane of lipid vesicles for at least 24 hours.

Summary

109

After demonstrating the stability of lipid-DNA in the liposomal bilayer,

lipid-DNA induced liposome fusion was studied in Chapter 3. The result

demonstrates the importance of the DNA-anchoring strategy in

hybridization-induced vesicle fusion, as not only the structural properties

of the unit itself, but also the number of anchoring units determine the

fusogenic properties. It was found that the orientation of DNA

hybridization and the number of anchoring units played a crucial role in

liposomal fusion. The zipper-orientated hybridization is more efficient than

non-zipper-orientated hybridization, which supports that double-stranded

DNA close to the vesicle surface could bring the docked vesicles in close

proximity to enhance full fusion. In the zipper orientation, the

hybridization event including vesicles with complementary sequences

brings their membranes in close proximity than in the non-zipper

configuration. Meanwhile, compared to vesicles functionalized with single-

anchored or double-anchored DNA, liposomes containing quadruple-

anchored oligonucleotides were proved to be highly fusogenic, achieving

considerable full fusion of up to 29% without notable leakage, which might

be related to the higher affinity of a quadruple lipid anchor to the

membrane. With this fusion system the most efficient fusogenic DNA

probes were produced known to date.

To further extend the functionality of DNA-based vesicles, the study

presented in Chapter 4 was focused on the functionalization of DNA

amphiphiles in a phospholipid bilayer. We explored DNA hybridization and

the dynamic exchange of DNA sequences on the surface of liposomes by

simple addition of DNA sequences with two FRET systems. FRET between

C594 (acceptor) and U4T-18-grafted Rh-DHPE-containing (donor) vesicles

demonstrated that DNA hybridization was achieved on the surface of

liposome. Subsequently, a 20-mer DNA oligonucleotide was introduced to

replace U4T-18 from C594 due to full hybridization. The disassembly of

C594 from the vesicle surface was realized since C594 was attached to the

liposome employing 14 complementary nucleotides while the removal

strand formed a duplex involving 20 nucleotides. The hybridization energy

for the latter is larger than for a 14mer duplex structure. Afterwards, the

free U4T-18 hybridized with C488, which is a donor for Rh-DHPE-

containing vesicles. Moreover, a DNA based amplification process was

Summary

110

performed on the surface of liposomes with a DNA probe, M2-Cy3. A

remarkable fluorescence intensity of Cy3 was obtained due to the DNA

hybridization chain reaction, which confirmed the capability of the

multiplication of surface functionalities from a single DNA anchoring unit

on the vesicle surface.

At the end of the thesis, a more complex membrane system was

functionalized with DNA. In Chapter 5, the incorporation of nucleobase

quadruple-anchored DNA in the surface layer of zebrafish embryos was

evaluated. The payloads connected by the supramolecular tether DNA can

be reversibly removed employing a removal strand, which represents a

very mild stimulus just requiring the addition of a DNA sequence not

affecting the life of the fish. Similar as on the vesicle surface, we

successfully demonstrated the performance of a DNA mediated

amplification process on the fish skin. The hybridization chain reaction

allows attachment of multiple moieties on a single anchored DNA strand

allowing multiplication of cargoes or signals on the surface. Moreover, it

was shown that surface modification of model membranes in form of

liposomes by various DNA nanotechnology procedures could be easily

transferred to the live animal. This allows establishment of DNA based

surface functionalization procedures and their facile and fast

implementation in zebrafish. Due to the broad application of zebrafish as

animal model in drug development, toxicology and nanoparticles

characterization in living systems, we believe the platform presented here

allows amalgamation of DNA nanotechnology tools with live animals and

enables efficient bio-barcoding as well as in vivo tracking.

Overall, this thesis has shown that chemical synthesis is a valuable tool to

produce functional DNA molecules to increase the complexity of membrane

engineering approaches. This was achieved by modification of nucleobases

by hydrophobic units and solid phase synthesis to fabricate amphiphilic

DNA strands. The piercing of hydrophobic units on the DNA into the inner

part of phospholipid membranes leads to stable anchoring. With this as a

starting point, the dynamic process of vesicle fusion was achieved, which

might be important in the future for synthesizing minute amount of

compounds by exploiting content mixing of liposomes filled with different

Summary

111

reactants. Similarly, the fusion approach might be utilized in the context of

actively transporting vesicle payloads in cells. In this case, the DNA

hybridization will fuel the fusion of vesicle with cell membranes to deliver

cargoes in the cytosol without being dependent on endocytosis processes.

One could even think of transferring this concept to live animals as

demonstrated in this thesis for zebrafish. It is without any doubt that DNA

amphiphiles bear lots of future potential for sophisticated DNA

nanotechnology functions in the realm of synthesis biology.

Samenvatting

Samenvatting

114

Samenvatting

Grote inzet is toegewijd aan het gebruik van DNA als bouwsteen in de

nanotechnologie door zijn veelzijdige eigenschappen, zoals hoge

specificiteit en programmeerbaarheid om complexe structuren te vormen.

De snelle ontwikkeling van chemische methodologie is bewezen nuttig te

zijn, niet alleen voor de bereiding van ongerepte DNA en RNA, maar ook om

basen- of ruggengraatmodificaties van de oligonucleotiden evenals vele

analogen te maken. De structurele verscheidenheid is verder uitgebreid

van 2D-patronen of 3D-nano-objecten, welke hoofdzakelijk zijn

vervaardigd met ongerept DNA, naar DNA-hybriden, zoals hybriden van

DNA-nanodeeltjes waaronder anorganische of zachte materialen. In het

bijzonder is de fosforamidiet-chemie aangepast voor de modificatie van

DNA met hydrofobe polymeren of lipiden, waardoor DNA-amphiphiles

worden verkregen. Deze DNA-amphiphiles kunnen worden gebruikt om

oppervlakte modificaties op biologische membranen uit te voeren.

In Hoofdstuk 2 hebben we een nieuwe strategie vastgesteld voor het

verankeren van oligonucleotiden in vesikelmembranen door middel van

hydrofobe eenheden aan de nucleobase te bevestigen. De membraanankers

worden in de oligonucleotide geïncorporeerd door geautomatiseerde

vastefasesynthese waardoor nauwkeurige controle over de positie en het

aantal hydrofobe eenheden binnen een DNA-sequentie mogelijk is. Een

FRET-systeem werd gebruikt om de integratie en stabiliteit van lipide-DNA

op het liposomale membraan te bewijzen. Enkelstrengs DNA

Samenvatting

115

gefunctionaliseerd met vier lipide-gemodificeerde nucleobasen was

gedurende ten minste 24 uur stabiel geënt op het membraan van

liposomen.

Nadat de stabiliteit van lipide-DNA in de liposomale dubbellaag was

aangetoond, werd lipide-DNA geïnduceerde liposoomfusie bestudeerd in

Hoofdstuk 3. Het resultaat laat het belang zien van de DNA-

verankeringsstrategie bij hybridisatie geïnduceerde vesikelfusie, aangezien

niet alleen de structurele eigenschappen van de eenheid zelf, maar ook het

aantal verankeringseenheden de fusogene eigenschappen bepalen. Er werd

gevonden dat de oriëntatie van DNA hybridisatie en het aantal

verankeringseenheden een cruciale rol speelden bij liposomale fusie. De

rits-georiënteerde hybridisatie is efficiënter dan de niet-rits georiënteerde

hybridisatie, wat ondersteunt dat dubbelstrengs DNA vlakbij het

vesikeloppervlak de verbonden vesikels in nabijheid zou kunnen brengen

om volledige fusie te verbeteren. In de rits-oriëntatie brengt de

hybridiseringsgebeurtenis vesikels met complementaire sequenties in hun

membranen dichter bij elkaar dan in de non-ritsconfiguratie. In

tegenstelling tot vesikels welke gefunctionaliseerd zijn met enkelvoudig

verankerd of dubbel verankerd DNA, bleken liposomen welke viervoudig-

verankerde oligonucleotiden bevatten zeer fusogeen zijn, waardoor een

aanzienlijke volledige fusie tot 29% werd bereikt zonder opmerkelijke

lekkage, wat mogelijk verband houdt met de hogere affiniteit van een

viervoudige-lipide anker in het membraan. Met dit fusiesysteem werden de

meest efficiënte fusogene DNA probes tot nu toe geproduceerd.

Om de functionaliteit van DNA-gebaseerde vesikels verder uit te breiden,

was de studie in Hoofdstuk 4 gericht op de functionalisatie van DNA-

amphiphiles in een fosfolipide dubbellaag. We onderzochten DNA

hybridisatie en de dynamische uitwisseling van DNA sequenties op het

oppervlak van liposomen door middel van de toevoeging van DNA

sequenties met twee FRET systemen. FRET tussen C594 (acceptor) en

U4T18-geënte Rh-DHPE-bevattende (donor) vesikels toonde aan dat DNA

hybridisatie op het liposoomoppervlak werd bereikt. Vervolgens werd een

20-mer DNA oligonucleotide geïntroduceerd om U4T18 van C594 te

vervangen door volledige hybridisatie. De demontage van C594 uit het

Samenvatting

116

vesikeloppervlak werd gerealiseerd aangezien C594 aan het liposoom was

verbonden met 14 complementaire nucleotiden, terwijl de

verwijderingsstreng een duplex vormde waarbij 20 nucleotiden betrokken

waren. De hybridisatie-energie voor deze laatste is groter dan voor een 14-

mer duplexstructuur. Daarna werd de vrije U4T18 gehybridiseerd met

C488, welke een donor is voor Rh-DHPE-bevattende vesikels. Verder werd

een DNA-gebaseerd amplificatieproces uitgevoerd op het oppervlak van

liposomen met een DNA-probe, M2-Cy3. Een opmerkelijke fluorescentie-

intensiteit van Cy3 werd verkregen door de DNA-hybridisatie ketenreactie,

welke de mogelijkheid van vermenigvuldiging van oppervlaktefuncties uit

een enkele DNA-verankeringseenheid op het vesikeloppervlak bevestigde.

Aan het eind van het proefschrift werd een complexer membraansysteem

gefunctionaliseerd met DNA. In hoofdstuk 5 werd de opname van

nucleobase viervoudig-verankerd DNA in de oppervlaktelaag van zebravis

embryo's geëvalueerd. De lading verbonden door de supramoleculaire

DNA-ketting kunnen omkeerbaar verwijderd worden door gebruik te

maken van een verwijderingsstreng, welke door enkel de toevoeging van

een DNA-sequentie een zeer milde stimulus vertegenwoordigt wat de

levensduur van de vis niet beïnvloedt. Net als op het vesikeloppervlak,

hebben we met succes de prestatie van een DNA gemedieerde

amplificatieproces op de vissenhuid aangetoond. De

hybridisatieketenreactie maakt het mogelijk om meerdere delen op een

enkele verankerde DNA-streng toe te voegen, waardoor vermenigvuldiging

van ladingen of signalen op het oppervlak mogelijk is. Bovendien toonde

het aan dat oppervlakmodificatie door verschillende DNA-nanotechnologie

procedures aan modelmembranen in de vorm van liposomen gemakkelijk

op het levende dier kon worden overgedragen. Dit maakt het mogelijk om

DNA-gebaseerde oppervlakfunctionalisatie procedures en hun eenvoudige

en snelle implementatie in zebravis te bepalen. Door de brede toepassing

van zebravis als diermodel in de ontwikkeling van geneesmiddelen,

toxicologie en nanodeeltjes karakterisatie in levende systemen, geloven wij

dat het hier gepresenteerde platform de combinatie van DNA-

nanotechnologie met levende dieren in staat stelt en efficiënte bio-

barcoding en in-vivo tracking mogelijk maakt.

Samenvatting

117

Globaal gezien heeft dit proefschrift aangetoond dat chemische synthese

een waardevol hulpmiddel is om functionele DNA-moleculen te produceren

om de complexiteit van membraan aanpassingsbenaderingen te verhogen.

Dit werd bereikt door modificatie van nucleobasen door hydrofobe

eenheden en vastefase synthese om amfifiele DNA strengen te fabriceren.

Het doordringen van hydrofobe eenheden op het DNA in het binnenste

gedeelte van fosfolipide membranen leidt tot stabiele verankering. Hiermee

werd het dynamische proces van vesikelfusie bereikt, wat in de toekomst

belangrijk kan zijn voor het synthetiseren van kleine hoeveelheden door

het gebruik van de inhoudsmenging van liposomen gevuld met

verschillende reactanten te exploiteren. Op dezelfde manier zou de

fusiebenadering gebruikt kunnen worden in de context van het actief

transporteren van beladen vesikels in cellen. In dit geval zal de DNA

hybridisatie leiden tot de fusie van vesikels met celmembranen om

ladingen in het cytosol te leveren zonder afhankelijk te zijn van

endocytische processen. Men zou zelfs kunnen denken over het

overbrengen van dit concept op levende dieren, zoals reeds aangetoond in

dit proefschrift voor zebravissen. Het is zonder twijfel dat DNA-amfifielen

een groot toekomstpotentieel hebben voor geavanceerde DNA-

nanotechnologie functies op het gebied van synthetische biologie.

Acknowledgements

Acknowledgements

120

Acknowledgements

So happy to finish this thesis! 2017 has been the most important year to me

up to now. This is not only because I finished my PhD study in Groningen

University but also a lovely new member joined our family, my lovely

daughter Ruby. Looking back to the past five years, I am filled with all sorts

of feelings and memories. It would not be possible to finish this thesis

without the help of many kind people surrounding me. I would like to

gratefully acknowledge those who have contributed to this thesis and

supported me during my PhD study.

First and foremost, I would like to give my deepest gratitude to my

supervisor, Prof. Andreas Herrmann, who gave me the opportunity to

conduct PhD study in Netherlands and be a part of our group! I still

remember how excited I was when I received the offer letter from you. I

learned a lot from the discussion with you in your office, where you always

patiently explained the ideas and mechanisms to me. I learned a lot from

your broad knowledge, inspiring ideas and enthusiasm in scientific

researches. Also, I would like to thank you for your encouragement which

was supportive for my first two years when I was painful and lost because

of the failing results. And I also remember how delighted and relieved I was

when I finally got the fruitful time and finished all the manuscripts. Those

experiences trained me to be a person with independent thought and an

open mind which is helpful for my future study and work. Also thank you

Acknowledgements

121

so much for offering me the researcher contract when I was pregnant,

which was a tremendous help to our life and study.

I also would like to thank the members of the reading committee: Prof. S.

Vogel, Prof. A. M. van Oijen, and Prof. D. J. Slotboom for the time and the

evaluation on my thesis. I want to express my special thanks to you for

your valuable comments which helped me improve the manuscript.

I am deeply grateful to Prof. Alexander Kros in Leiden University. It was a

great experience and fruitful collaboration with your group. Thank you

very for your patience and dedicated time on the experimental design, data

analysis and paper writing. At the meantime, I would like to thank your

group member: Dr. Jian Yang. Your great efforts helped me on my work

and I learned a lot from the work together with you.

I would like to thank other collaborators involved in my projects. Prof. B.

Poolman, Prof. A. Kocer, Prof. S. J. Marrink, Prof. A.M. van Oijen, Dr.

Duygu, Dr. Gemma and Rianne. I am really grateful to all of you for the

valuable discussions on my projects, which helped a lot to improve my

scientific work. I want to thank Jelle for dedicated time to do TIRFM

measurements together with me. The discussions with you are always

inspiring and joyful.

Also, I take this opportunity to thank China Scholarship Council (CSC).

Thanks for the scholarship for my four years’ PhD study, which gives me

the opportunity to go abroad and study. Meanwhile, I would like to show

my gratitude to my Alma mater, Zhengzhou University (ZZU), which gave

me full support when I applied for the CSC scholarship. So far as I know,

ZZU is the only University who establishes a free English course for CSC

applicants and pays for their IELTS or TOEFL test fees. To help us better

understand and communicate with foreign universities, ZZU encouraged

and subsidized us to go to Beijing to join the CSC meeting. When I went

abroad and started my PhD study, I got a suitcase form ZZU like a gift which

is full of encouragement and expectation. Thanks to Prof. K. Y. Tang and

Prof. X. J. Zheng in Zhengzhou University. Both of you gave me great

support during my application of CSC scholarship and allowed me to have

my Master defense ahead of schedule, which made my PhD study abroad

Acknowledgements

122

possible. I am very grateful to both of you for your encouragement and

comfort when I felt confused and loss about my PhD study during the first

two years.

Thanks to everyone in the PCBE group for being always nice and helpful.

Very special thanks to Bart for your valuable discussions and dedicated

time on my manuscript polishing. Karin, many thanks to you for your help

with all the paper work. I remember last time I gave you my claim for the

stay in Leiden but found two receipts were lost. I was going to pay by

myself since it was just 30 euro. But you told me that a student shouldn’t

pay for that and you would help me argue for it if the finance department

questioned me, which made me feel fully supported! Ursula, thanks so

much for your efforts on the submission of my thesis. Without your help, it

cannot go so smooth! And you are so efficient and enthusiastic. Every time

I go to your office and ask if you can help me do something, you always

gave me positive answers! Wish you will have a wonderful stay in China!

Special thanks to Evgeny for your invaluable technical support on my

project.

Special thanks are given to Alberto, my first daily supervisor. Thanks for

your patience and understanding with me when my English was so poor to

communicate. Your Spanish enthusiasm made me feel warmly welcomed

and thanks so much for teaching me everything, not only the experimental

operation, but also the data analysis and PowerPoint design during my first

days in our lab. And also thank you and your wife for your warm hospitality

when Qing and I were in Madrid. Agnieszka, you are a “walking heater” to

me. When I was lost and had no idea about writing the introduction of my

thesis, you helped me clarify my thoughts and pinpoint the right direction.

You gave so much help to my experiments and my personal life. Thanks for

your delicious Polish food, the cute sweater to my little Ruby and the

wonderful “girls’ night” in your sweet apartment. Jan Willem, you are

always positive, smiling and helping everyone. You took me to the stationer

to get all the stationery and taught me how to use the instruments in our

lab. Whenever I got a problem and came to you, you always sat down and

answered my questions. Alina, so many thanks for your helping my

Acknowledgements

123

experiments and you showed me a different perspective to look into the life.

I remember when I told you I didn’t want to use EtBr because of its toxicity

and you said you can do it for me if I was so worried. To be honest, I was

shocked because I would never do something like that. I really enjoyed all

the fun time with you in and out of the lab, like the first time for me to have

a relaxing time in a hot spring and we went to the painting shop to find a

dyestuff to cover the flaw on my door. Thanks also to Alessio and Diego for

your discussions and fun time in the lab and office. Dear Mark, thanks a lot

for your efforts on my thesis and your kindly help in Qing’s and my life, like

making a Dutch phone call or reading a Dutch letter. Wish you find a

girlfriend ASAP! Eliza, it’s so nice to have you in life! You gave me so many

advices when I needed your help like our trip to Poland. We had a

wonderful journey with your detailed suggestions! Hongyan, thanks very

much to be my Paranymph and it’s surprisingly good to live together with

you for the last two months! Wish you will have more high quality papers

and find your soulmate! Wei, Jun & Pei, Avishek and Pavlo, you are

always willing to help me in my experiments.

My great thanks go to Jingyi for all your warm help during my five years

life in Groningen. Whenever I meet a problem, you are the first person I

prefer to ask for help because I know I will get detailed suggestions and

comfort from you. Countless delicious dishes and food were taken in your

apartment with your excellent cooking skill. I would never forget the good

time when you, Shuo, Qing and I watched the Chinese show in your giant

TV and enjoyed the hotpot in the cold winters. I always feel the quality of

my life was reduced since you got pregnant… I wish Shou & you all the best

and every success! Lei, I really appreciate your efforts on our collaborated

experiments. You are open to share your working experiences and it’s very

nice to work together with you. There is so much fun time with you in and

out of the lab, like we went to Paris for holiday together. We were worried

to get robbed in Sacré-Cœur and discussed so many notes on security. And

finally found we were over-worried and had a nice time there. Wish

Zhongtao and your work went well in China.

I sincerely like to express my appreciations to Lifei and Kai. Both of you are

like “second professors” to me. Lifei, your rich specialty base and multi

Acknowledgements

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discipline knowledge always amazed me when I discussed with you. Your

analysis capability is so brilliant that you gave me lots experiment

suggestions even the project had no relation with yours. I’ll treasure the

good time that we shared one apartment and lived together. Your excellent

cooking skill gave me lots of wonderful “delicious” memories. Qing and I

will be your first customers if your noodle restaurant was open, which

however would be a huge loss for science world. May Qinhong, Lulu & you

live a happy life! Kai, I really appreciate your advice, the fruitful

discussions and our collaborations. Your limitless compassion for study

and experiment always remind me that I can do better. You are like a “gold

digger” that you can find flashpoints from a lot experimental results which

is dependent on your diligent literature reading. Without you, the studies

could not be performed, and we could not have such nice publications.

Wish Juanjuan & you have a good time in USA.

Jing, I really appreciate so many pleasant times with you and Jiaying, like

we went to Keukenhof to enjoy the beauty of tulips and picked up the

Chinese chestnut when I was pregnant. Also there were lots of good

memories of our trip to Portugal and we were a good team. I was the guide.

Qing was the bodyguard. Jiaying was the map. You were a very competent

sentry since you had such a high level of vigilance. And thanks very much

for the nice food and taking care of us when we came back to Groningen

after a long train travel. Yu, Pengkun, Shuaidong, Miancheng, Xintong,

Gurudas, Karolin and Kseniya thanks very much for being my colleagues

in PCBE group. It is indeed a very wonderful experience to work together

with all of you.

Dear friends, Qiuyan, thank you so much for your kindly help during my

life in Groningen. I was so afraid and nervous when I the first time arrived

in Schiphol airport. It was so nice of you to take a 3 hours train to Schiphol

to pick me up which was a big relief to me. You took me to the supermarket

and helped me to get my first bicycle in Groningen which made my first day

here much easier. You always asked me to go outings with you and your

friends, which made me not feel lonely. What impressed me most was

when you knew I was sad and crying for the death of my hamster, you came

to comfort me and buried my hamster together with me. You are really a

Acknowledgements

125

good friend to me and I wish you all the best in the future! Tao & Wenjun, I

really enjoy the Majiang nights with you guys and hopefully we can do it

again in the future. Yu, thanks so much for giving me advise and comfort

when I was scared of the delivery. And you always give me detailed

information when I ask you questions, like Ruby’s first rash. Wish June,

Hein & you a happy life! Tiancai & Yang, I am missing your yummy noodle

and looking forward to meeting you and your little girl at your defense. Jin,

thanks very much for your help when I was confused about my TEM results

and you are always patient to answer my questions. Chao & Xin, thanks

very much for taking care of dundun when Qing and I was on holiday. Wish

you all the best in the future! Guowei, thank you for giving me the

opportunity and trust to design your thesis cover. It’ll not be free next time

(*^__^*).

Meanwhile, I would like to express my appreciation to Groningen

University (RUG). RUG is a fantastic and amazing university, and I am so

fortunate to be part of it. RUG is at the forefront in providing freedom and

equality for all the students. When we first came here, we had

1200euro/month scholarship from CSC and found the other PhDs have

much higher salary than us. We felt kind of wronged since we did the same

work. So we wrote a joint letter and sent it to the headmaster nervously.

This was the first and only time in my life (until now) to sign a joint letter. I

felt quite perturbed and worried because I wasn’t taught to express my

demands. It’s inspiring that the university discussed over our letter in a

committee meeting and finally decided to give every CSC student 400euro

per month as a housing allowance. You cannot image how jealous they are

for CSC PhD from the other university. ↖(^ω^)↗

Also, thanks for the American sitcom, How I Met Your Mother, which

brings Qing and me so many joys and happiness. Although it is a comedy,

HIMYM taught us how to deal with failure, hold on to dreams, and even face

the death. There were countless times that we were tired and exhausted

after one day work, we watched HIMYM to get relaxed, like talking to some

old friends.

Acknowledgements

126

Finally, I would like to give my gratitude to my family. 首先感谢我的爸爸妈

妈，感谢您们这么多年对我学习和生活上的支持。每当我困惑失落的时候，

你们总能给我鼓励和支持。感谢黄妈妈，感谢您来荷兰帮我们照看小宝宝，

让我们可以放心的进行工作和学习。My little girl, Ruby 小梦安, I always

feel sweet and implausible when I look at you. Here is a quote from

“Spider-man” which could accurately represent my feeling to you. “When I

look in your eyes, and you’re looking back in mine, everything feels not quite

normal. Because I feel stronger and weaker at the same time, I feel excited

and at the same time terrified. The truth is I don’t know what I feel, except I

know what kind of man I want to be.”

Qing, my dear husband and best friend. I am so lucky to meet you at the

right place and right time. Life in a foreign country is not easy. Without you

I cannot finish my PhD study. There were so many times that I was

confused, self-doubted and about to give up because of the endless and

hopeless failing experiment results, you always gave me tremendous

encouragement and support. Your patience, respect, tolerance and

understanding helped me be a better person. When we were traveling in

Hallstatt, we got trapped in the big snow and couldn’t find the hotel. It was

dark and cold in the night, but I felt so safe and warm because you were

there. You are like some kind of superhero that could solve any problem

and protect me. Life is like unknown journeys, and I expect the next one

together with you.

Publications

127

Publications

1. Z. Meng, J. Yang, Q. Liu, J. W. de Vries, A. Gruszka, A. Rodrίguez-Pulido, A.

Kros, A. Herrmann. Efficient Fusion of Liposomes by Nucleobase Quadruple-

Anchored DNA. Chem. Eur. J. 2017, 23, 9391-9396.

2. Z. Meng, Q. Liu, J. Sun, M. Loznik, K. Liu, A. Herrmann, Highly stiff and

stretchable DNA liquid crystalline organogels with fast self-healing and

magnetically responsive behaviors. Nat. Commun. Submitted.

3. J. Yang, Z. Meng, Q. Liu, Y. Shimada, R. C. L. Olsthoorn, H. Spaink, A.

Herrmann, A. Kros, Employing DNA hybridization for Zebrafish Surface

Engineering. Angew. Chem. Int. Ed. Submitted.

4. Z. Meng, K. Liu, Q. Liu, P. Zhao, A. Herrmann, Study of the Hybridization

Properties of DNA-Surfactant Complex in Organic Phase. Manuscript in

preparation.

5. L. Zhang, L. Zheng, Z. Meng, K. Balinin, M. Loznik, A. Herrmann, Accelerating

Chemical Reactions by Molecular Sledding. Chem. Commun. 2017, 53: 6331-

6334.

6. A. S. Lubbe, Q. Liu, J. W. de Vries, J. C.M. Kistemaker, A. H. de Vries, I. F. Plo, Z.

Meng, W. Szymanski, A. Herrmann and B. L. Feringa. Photoswitching of DNA

hybridization using a molecular motor. J. Am. Chem. Soc. Submitted.

7. Q. Liu, Z. Meng, H. Fang, H. Li, K. Liu, A. Herrmann, Fluorescence properties

of lipid-DNA in liquid and liquid crystal states. Manuscript in preparation.

8. Q. Liu, A. Gruszka, J. Hurst, J. W. de Vries, F. Fröß, U. Hage, Z. Meng, K. U. B.

Schmidt, S. Schnichels, A. Herrmann, M. Spitzer, Lipid modified aptamers as

vehicles for ophthalmic drug delivery. Controlled Release. Submitted.

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