RECOGNIZING METHYL-CPG SITES: INVESTIGATNG ZBTB PROTEIN REGULATION OF EPIGENTICALLY MODIFIED DNA

Albert M. Lund,1 Tommy W. Terooatea,1 Kuei-Ho Chen1, and Bethany A. Buck-Koehntop1*

1Department of Chemistry, University of Utah, 315 S. 1400 E., Rm. 2020, Salt Lake City, UT, USA

* Correspondence to: koehntop@chem.utah.edu

ABSTRACT

Methylation of cytosine in CpG dinucleotides leads to subsequent gene suppression and can result in cancer progression if misregulated. These epigenetic DNA mod-ifications and the factors that regulate them are attrac-tive cancer therapeutic targets since the damage is re-versible unlike in genomic mutations. Here we utilize a multi-disciplinary approach to investigate the role that the Zbtb family of methyl-CpG binding proteins (MBPs) plays in modulating these epigenetic signals in cancer.

INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION

In eukaryotes, methylation of CpG dinucleotides is es-sential for genomic stability, control of gene expression and the regulation of chromatin structure in normal cells. Aber-rant alterations in these genomic methylation patterns have a direct link to a variety of diseases, including cancer. The Zbtb family is a set of specialized transcription factors, which exhibit bimodal DNA recognition by specifically targeting both methylated DNA signals as well as sequence-specific non-methylated sites [1-3] through a set of three conserved Cys2His2 zinc fingers. DNA recognition subse-quently recruits chromatin remodelling machinery to the target site resulting in chromatin compaction and gene si-lencing.

While there are sufficient findings to suggest that the Zbtb family of MBPs participates in cancer progression, an extensive analysis of the gene targets and subsequent signal-ing pathways regulated by each protein has yet to be inves-tigated. Here we utilize an interdisciplinary approach com-bining structural biology, molecular biology and cellular

biology to determine the epigenetic signaling pathways reg-ulated by the Zbtb family of MBPs and to identify the pro-tein region(s) necessary for high-affinity DNA recognition.

We have designed multiple protein constructs around the conserved Cys2His2 zinc finger DNA binding region as well as additional zinc fingers located in the C-terminal regions of Zbtb4 and Zbtb38 as the functions of these additional zinc finger regions have not yet been identified. It is possi-ble that these regions also participate in DNA recognition and may be partly responsible for direct targeting of these proteins to differential gene targets. The constructs have been screened by HSQC analysis to determine whether they were amenable for further structural characterization and whether they exhibit DNA binding properties.

Additionally, using western blot analysis (Figure 1), we have determined that the three Zbtb proteins exhibit differ-ential endogenous expression levels in a variety of cancer cell lines. Of particular interest, all of these proteins were down-regulated in the indolent form of prostate cancer (LNCaP) but up-regulated in the aggressive form (PC3). Interestingly, the MBD family of MBPs exhibits the oppo-site trend between the indolent and aggressive forms [4]. Through ChIP-Seq and qPCR analysis, we have begun to catalog the gene occupancy of these proteins in prostate cancer cells.

In conclusion, our preliminary results indicate that the preferential gene targets in the PC3 cancer cells may define a new set of potential biomarkers that can be utilized to dis-tinguish aggressive from indolent forms of prostate cancer. Further, our initial structural analysis of the Zbtb proteins and their DNA interactions will provide the basis for a larg-er program in which atomic-level structural knowledge will be utilized for the design of novel and highly selective epi-genetic-based cancer therapeutics directed against the Zbtb family of MBPs.

REFERENCES

1. Daniel, J.M., Spring, C.M., Crawford, H.C., Reynolds, A.B., Baig, A. Nuc. Acid Res., 2002, 30, 2911-2919.

2. Fillion, G.J.P., Zhenilo, S., Salozhin, S. Yamada, D., Prokhortchouk, E., Defossez, P.-A. Mol. Cell. Biol., 2006, 26, 169-181.

3. Buck-Koehntop, B.A., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. FEBS Lett., 2012, 586, 734-739.

4. Pulukuri, S.M., Rao, J.S. Oncogene, 2006, 25, 4559-4572.

Figure 1. Western blot indicating the endogenous protein expres-sion levels for the three Zbtb family MBPs in various cancer cells.

154

ONE-THIRD-OF-THE-SITES BINDING OF TRANSITION-STATE ANALOGUES BY TRIMERIC

PURINE NUCLEOSIDE PHOSPHPORYLASES IN THE LIGHT OF NEW FINDINGS

Marta Narczyk,1 Beata Wielgus-Kutrowska,

1 Katarzyna Breer,

1 Mariko Hashimoto,

2

Sadao Hikishima,2 Tsutomu Yokomatsu

2 and Agnieszka Bzowska

1*

1Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Żwirki i Wigury 93, 02-089 Warsaw,

Poland and 2School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, To-

kyo 192-0392, Japan * Correspondence to: abzowska@biogeo.uw.edu.pl

ABSTRACT

One-third-of-the-sites binding of transition-state ana-

logues, immucillins was postulated to occur in the case of

trimeric purine nucleoside phosphorylases. We have

synthesized a new analogue with the PNP transition-

state features, 9-{[N-[3′′,3′′-difluoro-3′′-(diethylphospho-

no)propyl]amino]methyl}-9-deazaguanine, and showed

by calorimetric and fluorimetric titrations that it binds

similarly to all three sites of the enzyme. We give possi-

ble explanation why experiments with immucillin were

interpreted in terms of one-third-of-the-sites binding.

INTRODUCTION,

Purine nucleoside phosphorylases (PNP, E.C. 2.4.2.1)

from mammalian sources are homotrimeric enzymes, and

important drug targets for potential immunosuppressive and

anticancer agents. It was postulated that PNP exhibits one-

third-of-the-sites binding, it means one molecule of transi-

tion-state (TS) analogue inhibitors, immucillins, binds to

each PNP trimer and blocks binding to the remaining two

sites [1]. Later, on the basis of isothermal titration calorime-

try (ITC) it was suggested that binding to subsequent sites

occurs with negative cooperativity, and dissociation con-

stants (Kd) were estimated: 56 pM, 12 nM and 15 M [2].

RESULTS AND DISCUSSION

Immucillins have two features of the PNP TS: proton at

position N(7) of the purine base and positive charge on the

pentose ring imitating the oxocarbenium ion character of the

nucleoside in TS (Scheme, upper left panel). We have pre-

viously synthesized analogue with one TS feature (DFPP-

DG, Scheme, lower left panel), and reported that it binds as

tightly as immucillin (Kd = 85 pM) but shows no one-third-

of-the-sites binding [3]. However, when the recombinant

PNP is used in binding studies, the apparent cooperativity

may appear because some PNP active sites may be blocked

by hypoxanthine (PNP product), moped from the host E.

coli cells [3]. Here we present synthesis (Scheme, right pan-

el) of 9-{[N-[3′′,3′′-difluoro-3′′-(diethylphosphono)propyl]-

amino]methyl}-9-deazaguanine (aza-DFPP-DG) having

both features of the TS of PNP (Scheme, compound 6). By

ITC and fluorimetric titrations, spanning broad enzyme con-

centration range, we demonstrate (Figure 1) that binding of

aza-DFPP-DG with trimeric PNP is characterized by one

dissociation constant, and stoichiometry of the complex is

three ligand molecules per enzyme trimer. Hence, no one-

third-of-the-sites binding is observed for this TS analogue.

Supported by the Polish Ministry of Science and Higher

Education (grants N N301 044939 and BW-1724/BF).

REFERENCES 1. Miles, R.W., Tyler, P.C., Furneaux,R.H., Bagdassarian,

C.K., Schramm, V.L. Biochem., 1998, 37, 8615-8621.

2. Edwards, A. A., Mason, J. M., Clinch, K., Tyler, P. C.,

Evans, G. B., Schramm, V. L. Biochem., 2009, 48,

5226-5238.

3. Breer, K., Wielgus-Kutrowska, B., Girstun, A., Staroń,

K., Hashimoto, M., Hikishima, S., Yokomatsu, T.,

Bzowska, A. Biochem. Biophys. Res. Commun., 2010,

391, 1203-1209.

Figure 1. ITC and fluorimetric titrations of calf PNP by aza-DFPP-

DG at pH 7.0, 25oC. Concentration of PNP subunits is shown.

N

N

N

O

H

NH2

H

CF2PO3H2

HN

N

N

OPOM

N CHNMe2R

HN

N

N

OPOM

N CHNMe2

NH

CF2PO3Et2

5

HN

N

N

OH

NH2

NH

CF2PO3H2

6

12

R=IR=CHO

a

c

d

+ XCF2PO3Et2

3

4

X=N3X=NH2

bNH

OH OH

OH

N

N

N

O

H

H

1

23

4

56

78

9

(H+)

7.6 M

0.05 M

0.5 M

155

SYNTHESIS OF BRANCHED RNAS, LARIAT MIRTRONS AND RNA MINI-LARIATS AND THEIR

USE IN DEBRANCHING-DEPENDENT RNA INTERFERENCE

Subha R. Das,* Eduardo Paredes and Debasish Grahacharya

Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, Pittsburgh, PA 15213, USA. * Correspondence to: srdas@andrew.cmu.edu

ABSTRACT

The synthesis of branched RNAs as well as lariat

RNAs that include native phosphodiester or trizole

backbone linkages is described. These RNAs are sub-

strates for debranching enzyme and could find use in

RNA silencing by a non-canonical pathway.

INTRODUCTION, RESULTS AND DISCUSSION,

CONCLUSION

Backbone branched RNAs (bRNAs) are involved in the

distinct but related processes of splicing, debranching and

retrotransposition that are key to cellular regulation by RNA.

The first step of splicing – either within the spliceosomal

apparatus or in the group II intron – generates an RNA in

which an invariant adenosine branch-point residue is linked

at the 2'-position to the 5'-end of an RNA sequence that cor-

responds to the conserved residues of the intron. This bRNA

occurs as a ‘Y’-shaped structure in trans-splicing or as a

lariat structure, in which the sequence through the 2'-5'-

linkage loops around to include the branch-point adenosine,

in cis- or pre-mRNA splicing. Subsequently, the lariat or

bRNA is the substrate for lariat debranching enzyme (Dbr1),

a manganese dependent phosphodiesterase that is related to

well-described metallophosphatases such as calcineurin, λ

protein phosphatase and Mre11, a nuclease involved in

DNA repair[1,2]. Debranching of the 2',5'-phosphodiester

linkage in bRNA from splicing is also required for the pro-

cessing and biogenesis of box C/D snoRNAs7 and intronic

microRNAs (mirtrons) that bypass Drosha[3,4]. Additional-

ly, a lariat RNA form for Ty1 RNA has been proposed,

though the specific role of Dbr that is required for re-

trotransposition from Ty1 elements in yeast to HIV remains

unclear.

The significance of natural bRNAs and lariat RNAs in

biology has prompted significant efforts toward their syn-

thesis – since the first solution phase syntheses of tetranu-

cleotides by Caruthers and coworkers in 1986 and Chatto-

padhyaya and coworkers shortly after, to the pioneering and

seminal efforts of Damha and coworkers in solid-phase syn-

thesis, and more recent approaches of Wang and Silverman

using deoxyribozymes[5-8]. Although these methods have

yielded branched oligomers and lariat RNAs, the ability to

readily access natural and modified sequences that include

biochemically useful probes or functional group substitu-

tions, has remained elusive.

Here we describe our successful efforts in the solid-phase

synthesis of branched RNA. The use of an orthogonal 2'-O-

protecting group in an internal residue permits facile branch

synthesis. The synthesis protocol is robust and issues of

migration of the phospho-diester or -triester are addressed.

The method is versatile, overcoming previous limitations to

sequences or modifications. All four natural nucleobases

could be installed as the branch-point residue. Several

bRNAs that include alternate branch points or single atom

substitutions were synthesized for biochemical interrogation

of Dbr1. While Dbr1 shares high active site homology with

Mre1, a metallophosphatase that acts on DNA, our analyses

with the bRNAs reveal a Dbr1 substrate feature that distin-

guishes it for its specific role.

With the branching RNA synthesis developed, we could

enhance it with the use azide-alkyne click-chemistry – both

in solid phase and in solution on naïve RNA (with free 2'-

hydroxyls) using a pseudo-ligandless protocol. These fur-

nishe branched RNA in which the loop is closed to complete

the lariat structure. This chemical ligation yields a triazole

linked lariat RNA with the location of the triazole linkage

either at the branch-point 2'-position or downstream.

These synthetic lariats or mini-lariats are substrates for

debranching in vitro. We therefore tested them in a dual

luciferase reporter assay for in vivo RNA silencing and

compare their effectiveness compared to single stranded

RNA as well as duplex siRNA. These results will be pre-

sented.

REFERENCES

1. Chapman, K. B. & Boeke, J. D. Cell 1991, 65, 483-492.

2. Khalid, M. F., Damha, M. J., Shuman, S. & Schwer, B.

Nucleic Acids Res 2005, 33, 6349-6360.

3. Okamura, K., Hagen, J.W., Duan, H., Tyler, D.M., Lai,

E.C., Cell, 2007, 130, 89–100.

4. Ruby, J.G., Jan, C.H., Bartel, D.P. Nature 2007, 448,

83-86.

5. Kierzek, R., Kopp, D. W., Edmonds, M. & Caruthers,

M. H. Nucleic Acids Res. 1986, 14, 4751-4764.

6. Zhou, X.X., Nyilas, A., Chattopadhyaya, J. Nucleic

Acids Symp Ser. 1987, 18, 93-96.

7. Carriero, S. & Damha, M. J. Solid-Phase Synthesis of

Branched Oligonucleotides 2001 in Current Protocols

in Nucleic Acid Chemistry; John Wiley & Sons, Inc.

8. Wang, Y. M. & Silverman, S. K. J Am Chem Soc 2003,

125, 6880-6881.

156

Detection of DNA Modifications and Enzymatic Repair Activities Based on a Multiple Spin-Labelling Strategy coupled with PELDOR Analysis

Mélanie Flaender 1, Giuseppe Sicoli 1, Samia Aci-Seche 2, Thomas Reignier 1, Vincent Maurel 1, Christine Saint-Pierre 1, Yves Boulard 2, Serge Gambarelli 1, Didier Gasparutto 1,*

1 SCIB - UMR E3 CEA / UJF Grenoble 1, INAC, CEA Grenoble, 38054 Grenoble Cedex 9, France 2 SBIGEM - IBITEC, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France

* Correspondence to: didier.gasparutto@cea.fr

ABSTRACT

Spin-labeled oligonucleotides produced by click chemis-try can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labelling strategy with damaged DNA. Extensive and accurate analysis of DNA structure and enzymatic repair processes were per-formed after digestion by an AP-endonuclease.

INTRODUCTION

Pulsed ELDOR (pulsed electron–electron double reso-nance) is a method of choice to measure distances in various double spin-labelled biological systems [1, 2]. Recently, this method was used to study the structure of normal and modi-fied DNA.[3-9]. By developing a “click chemistry” ap-proach to efficiently labels oligonucleotides with high yields, it is therefore possible to develop more complex la-belling scenarii involving at least three spin-label positions. Through triangulation measurements, these could provide more information on the DNA structure and can be used to study DNA-protein interactions. RESULTS AND DISCUSSION

In the present work we demonstrate that it is possible to efficiently synthesize and study such a sophisticated and informative structural probe, to study specific DNA damage and its enzymatic repair [10]. The results obtained confirm that it is possible to prepare duplex DNA constructs contain-ing three spin labels and one abasic site type modification with close to normal secondary structure. The nitroxide la-bels introduced by a “click-chemistry”-based strategy do not prevent recognition of the abasic site analogue by DNA re-pair systems, such as the enzyme Endo IV, and have no ef-fect on its cleavage activity (Scheme). In addition, we were able to effectively measure individual distances and the pre-cise widths of the distributions in the triangle formed by the three spin-labels. The current spin-labelling approach is easy to implement and provides nitroxide-containing mole-cules with relatively narrow distance distributions, making it possible to develop complex labelling strategies for the de-tection of precise biological events. This demonstrates the potential of our approach for the study of modified DNA structures and DNA-protein interactions. Acknowledgements: This work was funded by the ANR (Blanc-0064-01).

REFERENCES

1. Schiemann, O., Prisner, T.F. Q. Rev. Biophys. 2007.

2. Jeschke, G., Polyhach, Y. Phys. Chem. Chem. Phys. 2007.

3. Schiemann, O., Piton, N., Mu, Y., Stock, G., Engels, J.W., Prisner, T.F. J. Am. Chem. Soc. 2004.

4. Sicoli, G., Mathis, G., Aci-Seche, S., Saint-Pierre, C., Boulard, Y., Gasparutto, D., Gambarelli, S. Nucleic Ac-ids Res. 2009.

5. Sicoli, G., Mathis, G., Delalande, O., Boulard, Y., Gas-parutto, D., Gambarelli, S. Angew. Chem. Int. Ed. 2008.

6. Schiemann, O., Cekan, P., Margraf, D., Prisner, T.F., Sigurdsson, S.T. Angew. Chem. Int. Ed. 2009.

7. Marko, A., Margraf, D., Cekan, P., Sigurdsson, S.T., Schiemann, O., Prisner, T.F. Phys. Rev. 2010.

8. Singh, V., Azarkh, M., Exner, T.E., Hartig, J.S., Drescher, M. Angew. Chem. Int. Ed. 2009.

9. Romainczyk, O., Endeward, B., Prisner, T.F., Engels, J.W. Mol. Biosyst. 2011.

10. Flaender, M., Sicoli, G., Aci-Seche, S., Reignier, T., Maurel, V., Saint-Pierre, C., Boulard, Y., Gambarelli, S., Gasparutto, D. ChemBioChem 2011.

Scheme. PELDOR analysis of DNA lesion excision using an original triple-spin labeled probe

157

CHALLENGING THE ROLE OF ELECTROSTATICS IN PNA BINDING: NEGATIVE CHARGE CAN INCREASE DUPLEX STABILITY WITH DNA AND RNA

Jennifer M. Heemstra1* and N. Tilani S. DeCosta1

1Department of Chemistry and the Center for Cell and Genome Science, University of Utah, 315 S 1400 E, Salt Lake City, Utah 84112, United States. * Correspondence to: heemstra@chem.utah.edu

ABSTRACT

The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes has largely been attributed to the lack of electrostatic repulsion between the uncharged PNA backbone and the negatively charged DNA or RNA backbone. However, there are no previously reported studies that rigorously test this hypothesis. Here we re-port that under medium to high salt conditions, nega-tively charged PNA actually binds more strongly to DNA and RNA than does positively charged PNA, providing surprising new insight into the role of electrostatics in PNA binding.

INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION

Peptide nucleic acid (PNA)1 is an artificial nucleic acid having unique physicochemical properties, which can large-ly be attributed to the fact that PNA has an achiral, peptide-like N-(2-aminoethyl)glycine backbone in place of the sug-ar-phosphate backbone found in DNA and RNA. PNA shows tremendous potential for use in molecular diagnostics and antisense therapeutics due to its greater binding affinity, selectivity, and strand-invasion capability relative to native nucleic acids, as well as its resistance to degradation by nu-cleases and proteases.2 The enhanced thermodynamic stabil-ity of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the un-charged PNA backbone and negatively charged DNA or RNA backbone.3 However, studies to establish or refute this key hypothesis have yet to be reported.

Here we investigate the role of electrostatics in PNA binding by synthesizing PNA strands (H-GTAGATCACT-NH2) having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. We have found that positively charged PNA displays negative salt dependence and nega-tively charged PNA displays positive salt dependence. Thus, at NaCl concentrations above 250 mM for PNA:DNA and 100 mM for PNA:RNA, duplexes containing negatively charged PNA are actually more thermally stable than du-plexes containing positively charged PNA.

These results directly challenge the long-held hypothesis that PNA duplex stability results from lack of electrostatic repulsion. Rather, the enhanced binding affinity of PNA is likely due to steric interactions and backbone preorganiza-

tion. The ability to incorporate negative charge without sac-rificing binding affinity is anticipated to enable the devel-opment of PNA diagnostics and therapeutics that take ad-vantage of both the inherent benefits of PNA and the multi-tude of delivery technologies recently developed for DNA and RNA.

REFERENCES

1. Nielsen, P., Egholm, M., Berg, R., Buchardt, O. Science 1991, 254, 1497-1500.

2. Peptide Nucleic Acids: Protocols and Applications; Nielsen, P. E., Ed.; Horizon Bioscience: United King-dom, 2004.

3. Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden, B., Nielsen, P. E. Nature 1993, 365, 566-568.

Figure 1. Tm vs [NaCl] for duplexes of (a) DNA and (b) RNA with PNA having no charge (PNA nf), three negative charges (PNA 3neg) or 3 positive charges (PNA 3pos). Conditions: 3 μM PNA, 3 μM RNA, 10 mM NaPi buffer with added NaCl, pH 7.2.

158

SYNTHESIS AND CHARACTERIZATION OF BRANCHED RNA THAT MIMIC LARIAT RNA

SPLICING INTERMEDIATES

Adam Katolik,1 Richard Johnsson,

1 David Sabatino,

1 Jeremy G. Lackey,

1 Eric Montemayor,

2

P. John Hart2 and Masad J. Damha

1*

1McGill University, 801 Sherbrooke St. W. Montréal, Québec H3A OB8,

2University of Texas Health Science Center,

7703 Floyd Curl Dr., San Antonio TX 78229. * Correspondence to: Email address masad.damha@mcgill.ca

ABSTRACT

We have developed new methods for the synthesis of

branched RNAs that mimic intronic lariat RNA inter-

mediates. These methods produce branched oligoribo-

nucleotide sequences of arbitrary length, base composi-

tion, and regiochemistry at the branch-point junction.

Lariat RNAs are intronic ‘lasso’-like structures whose 5′

ends are connected to an internal adenosine unit via a 2′,5′-

phosphodiester linkage. This linkage must be hydrolyzed by

the intron debranching enzyme (Dbr1) before a spliced in-

tron can be metabolized or processed into essential cellular

factors such as snoRNA and miRNA. Over the past several

years, our group has been synthesizing branched RNA

(bRNA) fragments as model systems for studying lariat

RNA land the structural basis of their recognition by dDbr1.

Much effort has been put forth to find a practical ways to

chemically synthesize bRNA via the solid-phase method.

Our first such approach utilized a 2′,3′-bis-O-

phosphoramidite to couple to two support-bound RNA

chains.[1] This method generated bRNAs with identical

sequences at the 2′ and 3′ positions of the branchpoint. Fol-

low up work focused on divergent methods that produced

branched RNA-DNA sequences of arbitrary base composi-

tion, length, and orientation around the branch-point junc-

tion.[2] This was accomplished by orthogonal deprotection

at an internal TBDMS group and growing the DNA se-

quence in the 5′-to-3′ direction using commercially available

reverse DNA amidites.[2a]

The present work is an extension of our ‘divergent’

method and produces branched oligonucleotides consisting

entirely of RNA. The major features of this strategy are the

use of (a) branching monomers (1-3) bearing 2′-ALE, 2′-DMTr, and 2′-Lev orthogonal protecting groups and b) ‘re-

verse’ RNA 2′-TBDMS 5′-phosphoramidites 4

(ChemGenes). The divergent, regiospecific methods for

constructing bRNA oligomers on controlled pore glass

(LCAA-CPG) are illustrated in Figure 1. Best results were

obtained when Method A and B were used in conjunction

with monomers 1 and 2, respectively. For example, when

Method A is used, the first arm (N4N3XN2N1) is constructed

in the 3′-to-5′ direction using commercially available 2′-TBDMS 3′-amidite monomers and branching synthon 1.

The cyanoethyl phosphate groups and 2′-ALE are removed

by treatment with (a) NEt3/MeCN at r.t. for 90 min; (b)

washing the support; and (c) treatment with 0.5 M hydrazine

hydrate, py/AcOH, r.t., 30 min. The second arm of the Y-

shape RNA is grown by adding a concentrated (0.3M) solu-

tion of 4 (N5) and continuing growing in the reverse 5’-to-3’

direction using 0.15M solutions of monomers 4.

Method C is analogous to that introduced in 1997 by Sproat

and co-workers [3], except that instead of their 2′-Lev, 3′-Pixyl 5′-amidite monomer [3], we employed the analogous 3

(i.e., 3′-DMTr instead of 3′-Pixyl). In our hands, removal

of the 3′-DMTr group causes 2-5% migration of the 3′-Lev

group to the vicinal 2′-OH position, resulting in the isolation

of the bRNA as regioisomeric mixtures. All bRNA se-

quences were characterized by MS and debranching assays

with Dbr1.

REFERENCES 1. Damha, M. J., Ganeshan, K., Hudson, R. H. Zabarylo S. V.

Nucleic Acids Research, 1992, 20(24), 6565-73.

2. (a) Braich, R.S., Damha, M.J. Bioconjugate Chemistry, 1997,

8(3), 370-377; (b) Damha, M.J. and Braich, R.S. Tetrahedron

Letters, 1998, 39, 3907-3910.

3. Grøtli, M., Eritja, R., Sproat, B, Tetrahedron, 1997, 53(33),

11317-11346.

159

Computing DNA oligonucleotides hybridization enthalpy within molecular dynamics modeling

A.A. Lomzov*, Y.N. Vorobjev, D.V. Pyshnyi

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia; *Correspondence to: lomzov@niboch.nsc.ru

Abstract: Development of new derivatives and analogues of nucleic acids (NA) and

reliable prediction of their physico-chemical properties is important both in practice and

basic research. Significant progress in development of software and hardware has made the

in silico research widely used. The goal of this work is to analyze an applicability of the

molecular dynamics (MD) modeling for calculating oligonucleotide hybridization enthalpy.

Methods and Algorithms: The enthalpies of DNA duplex formation were determined as a

difference of the internal energy of double- and single-stranded states which were

calculated from MD trajectory computed with Amber 11 software (UCSF, USA).

Computations were performed on NVIDIA GTX580 and Intel i7-2600 hardware.

Results: To determine optimal parameters of modeling we have used Dickerson-Drew

dodecamer (DDD) with well characterized secondary structure and thermal stability. We

have varied temperature, heating protocol, and ion concentration in implicit and explicit

solvent and compared averaged structures with those experimentally obtained. Using

optimal parameters of modeling we have shown that hybridization enthalpy of DDD

correlates well with experimental and calculated one via nearest neighbor model enthalpies.

The use of GPU has speeded up the modeling of DDD in implicit solvent up to 60 times

and up to 30 in explicit solvent in comparison with the single node CPU.

To verify the MD predictive ability we have collected database of experimentally

determined thermodynamic parameters (enthalpy, entropy and Gibbs energy) of

hybridization of 272 oligodeoxyribonucleotides. The total energy of oligonucleotide and its

complex were calculated from 2 ns trajectories simulated with optimal parameters. The

RMSD of calculated and experimental enthalpies was 15%.

Conclusion: The results obtained show that MD modeling allows one to calculate enthalpy

of matched DNA duplexes with high accuracy.

Availability: An extension of this work is retrieval of parameters of MD modeling for

more accurate prediction DNA duplex thermal stability, including complexes with

perturbation of the regular structure, that could be used instead experimental research.

This research has been supported by Integration grant SB RAS (86), RFBR (10-04-01492-

а) and by MCB programs of RAS.

160

RNA X AND THE IMPACT OF SECONDARY STRUCTURE ON THE STABILITY OF RNA

PHOSPHOTRIESTER LINKAGES

Tuomas Lönnberg1*

1Department of Chemistry, University of Turku, FIN-20014, Turku, Finland * Correspondence to: tuanlo@utu.fi

ABSTRACT

An oligonucleotide incorporating a branching RNA

phosphotriester linkage has been synthesized and its

hydrolysis studied in the presence of various partly

complementary oligonucleotides. Certain structural mo-

tifs stabilize the reactive phosphotriester by more than

two orders of magnitude, suggesting a half-life of several

hours for the so-called RNA X structure, proposed to be

formed during the splicing of mRNA.

INTRODUCTION

It has been suggested that two small nuclear RNAs, viz.

U2 and U6 snRNA of the human spliceosome, form a phos-

photriester structure (the so-called RNA X) upon attack of

the 2´-OH function of adenosine 21 of U2 snRNA on the

A53pG54 phosphodiester bond of U6 snRNA [1]. Given the

facile nucleophilic attack of the neighboring 2’-OH function

on the phosphotriester group of ribonucleoside 3´-

dialkylphosphates [2], this finding is rather unexpected. In

the proposed RNA X structure, however, the putative phos-

photriester linkage is part of a four-way junction motif that

might offer stabilization by orienting the flanking 2´-OH

unfavorably for nucleophilic attack (Fig. 1A). The extent of

such stabilization could be determined by studying the hy-

drolysis of a phosphate-branched RNA oligonucleotide

model in the presence of appropriately designed partly com-

plementary oligonucleotides (Fig. 1B-E).

RESULTS AND DISCUSSION

The oligonucleotide model incorporating a branching

3´,3´,5´-phosphotriester linkage was synthesized by manual

5-benzylthiotetrazole-promoted coupling of an appropriately

protected dimeric phosphoramidite building block to the

terminal 5´-OH of a CPG-bound oligonucleotide. Orthogo-

nal protections (DMTr and levulinoyl) were used in the 5´-

OH groups of the dimeric building block to allow synthesis

of the two branches independently. One of the 2´-OH

groups flanking the scissile phosphotriester linkage was

protected with a tert-butyldithiomethyl group, the other res-

idues being 2´-O-methylated.

Hydrolysis of the phosphate-branched oligonucleotide

model (Fig. 1B) was carried out at 25 °C in 50 mM tris(2-

carboxyethyl)phosphine buffer. Under these reducing condi-

tions, the disulphide bond of the tert-butyldithiomethyl pro-

tection is rapidly cleaved. Subsequent hydrolysis of the re-

sulting thiohemiacetal yields a free 2´-OH group, which

then attacks the phosphorus atom of the phosphotriester

linkage. Decomposition of the pentacoordinate phophorane

intermediate thus formed takes place by fission of the P-O5´

or one of the P-O3´ bonds and an order of magnitude slower

than previously reported for the respective trinucleoside

phosphotriester lacking oligonucleotide arms. Complete

hybridization of the main chain of the model with another

oligonucleotide (Fig. 1C) further stabilizes the phos-

photriester linkage by an order of magnitude and structures

placing this scissile linkage in a kinked position (Fig. 1D

and E) are even more stable, albeit only moderately.

CONCLUSION

Secondary structure can considerably stabilize an RNA

phosphotriester linkage, suggesting that the proposed RNA

X species could have a half-life of at least several hours

under physiological conditions.

REFERENCES

1. a) Valadkhan, S., Manley, J. L. Nature, 2001, 413, 701-

707; b) Valadkhan, S., Manley, J. L. RNA, 2003, 9, 892-

904.

2. a) Kosonen, M., Seppänen, R., Wichmann, O.,

Lönnberg, H. J. Chem. Soc., Perkin Trans. 2, 1999,

2433-2439; b) Mikkola, S., Kosonen, M., Lönnberg, H.

Curr. Org. Chem., 2002, 6, 523-538; c) Lönnberg, T.,

Kiiski, J., Mikkola, S. Org. Biomol. Chem., 2005, 3,

1089-1096.

Figure 1. A phosphotriester linkage within RNA X (A) and var-ious oligonucleotide model assemblies (B – E).

161

STRUCTURAL STUDIES ON FLUORINE MODIFIED NUCLEIC ACIDS

Nerea Martín-Pintado1, Maryam Yahyaee

2, Glen Deleavey

2, Anna Aviñó

3, Jonathan K. Watts

2, Ramón

Campos4, Guillem Portella

5, Modesto Orozco

5, Ramon Eritja

3, Masad Damha

2, and Carlos González

1*

1Instituto de Química Física Rocasolano, CSIC, C/ Serrano, 119, 28006 Madrid, Spain

2Department of Chemistry, McGill University, Montreal, QC, H3A 0B8, Canada

3Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials

and Nanomedicine, E-08028 Barcelona, Spain 4Spectroscopy and NMR Unit, Structural and Computational Biology Programme, Spanish National Cancer Center

(CNIO), C. Melchor Fernández Almagro, 3, 28029, Madrid, Spain 5Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, Baldiri Reixac 10-12, E-

08028 Barcelona, Spain and Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Spain Department of Biochemistry. University of Barcelona. Diagonal 647, 08028 Barcelona, Spain

E-mail: nerea@iqfr.csic.es

ABSTRACT

In this communication we will present some of our

studies on fluorine-modified nucleic acids.

INTRODUCTION, RESULTS AND DISCUSSION,

CONCLUSION

Nucleic acids analogs containing 2’-fluoro-arabino (2’F-

ANA) and 2’-fluoro-ribose (2’F-RNA) are interesting

compounds for its potential applications in antisense and

interference RNA therapy. When 2’F-ANA hybridizes to

its complementary RNA, the resulting complex is a

substrate of the enzyme RNase H, which cleaves the

RNA strand of DNA/RNA hybrids, but is not active

against pure RNA duplexes. On the other hand, siRNAs

composed of combinations of 2’F-ANA and 2’F-RNA

can activate the RISC complex and elicit potent gene

silencing [1]. The favored conformations of these two

analogs are different, and combining 2’F-ANA and 2’F-

RNA led to reduce affinity relative to an RNA/RNA

duplex. Thus, the binding affinity at key regions of the

siRNA duplex could be tuned by changing the pattern of

incorporation of DNA-like and RNA-like nucleotides. By

combining 1H and

19F NMR spectroscopy, we have

determined the solution structure of several quimeric and

hybrid duplexes, which sequences combine different

patterns of 2’F-ANA and 2’F-RNA nucleotides [2]. In

this communication, we discuss the three-dimensional

structure of these duplexes, and compare them with the

structure of 2’F-ANA/RNA hybrids determined in a

previous study. We have also determined the structure of

a modified DNA/RNA hybrid duplex containing a 2’,2’-

difluoro-deoxycytidine nucleotide.

On the other hand, quadruplex structures have attracted

considerable attention during the last years. Chemical

modifications in this kind of structures help understand

their stability and structural properties and also have

potential applications in Biology and in Supramolecular

Chemistry. We will present here some of our recent NMR

studies on fluorine modified G-quadruplexes based on

telomeric sequences. Fluorine substitutions in the 2’-

position of the sugar (2’F-ANA and 2’F-RNA) stabilize

parallel propeller quadruplex conformations.

2′F-ANA 2′F-RNA

REFERENCES

1. Deleavey G. Watts J. Alain T. Robert F. Kalota A.

Aishwarya A. Pelletier J. Gewirt A. Sonenberg N.

Damha M. Nucleic Acids Research, 2010, 38, 4547-

4557

2. Watts JK, Martín-Pintado N, Gómez-Pinto I,

Schwartzentruber J, Portella G, Orozco M, Gonzalez

C and Damha MJ. Nucleic Acids Res, 2010, 38,

2498-2511.

162

SYNTHESIS OF MONO AND MULTIPLE CONJUGATED OLIGONUCLEOTIDES BY "CLICK

THIOL" CHEMISTRY AND COMBINATION WITH CUAAC "CLICK CHEMISTRY"

Albert Meyer, Jean-Jacques Vasseur and François Morvan*

Institut des Biomolécules Max Mousseron, UMR 5247 CNRS UM1 UM2, Université Montpellier 2, Place E. Bataillon, 34095 Montpellier cedex 5, France. *Correspondence to: morvan@univ-montp2.fr

ABSTRACT

Oligonucleotide conjugates were efficiently synthe-

sized by thiol click chemistry starting from a mono- or

poly-thiol oligonucleotides and different acrylamide de-

rivatives. This strategy was applied to form glycoclusters

and was also combined with CuAAC for bis-conjugation

through a sequential protocol.

INTRODUCTION

Oligonucleotide conjugates are widely used for various ap-

plications in biology, biotechnology, and medicine. Most

applications require labelling with dyes, redox tag or other

biomolecules such as biotin or carbohydrates.

We explored the use of thiol Michael-type addition (TMTA)

to prepare oligonucleotide conjugates. This addition corre-

sponds to the reaction of a thiol on an electron-deficient ene

forming a thioether linkage. This reaction is usually restrict-

ed to the reaction of thiol-oligonucleotides with maleimide

derivatives [1]. Herein we present the TMTA using acryla-

mide derivatives. Finally, TMTA was combined sequential-

ly with the Copper (I) catalyzed Azide Alkyne Cycloaddi-

tion (CuAAC) to synthesized bis conjugates.

RESULTS AND DISCUSSION

A 5'-S-acetyl-thiohexyl oligonucleotide was synthesized

using commercially available amidites on a DNA synthesiz-

er according to the phosphoramidite chemistry. Different

acrylamide derivatives exhibiting a phenyl, mannose, ferro-

cene, dansyl, biotin or deoxycholic moiety were prepared.

After the removal of cyanoethyl group by piperidine treat-

ment, the solid-supported thio-oligonucleotide was treated

with an acrylamide derivative in presence of TCEP and

K2CO3 methanol leading to the deprotection, release from

solid support and TMTA (Figure 1). Hence the monoconju-

gate oligonucleotide was obtained with a quantitative con-

version of the thiol-oligonucleotide. A size exclusion chro-

matography on cartridge allowed the isolation of almost

pure conjugate. The same strategy was applied to synthesize

a mannose-centered tetramannose oligonucleotide allowing

the formation of a glycocluster oligonucleotide conjugate

with high efficiency starting from a tetra-thiolhexyl oligo-

nucleotide.

Finally the TMTA was combined with the CuAAC ac-

cording to a sequentially protocol to synthesize bis-

conjugated oligonucleotides exhibiting biotin and carbohy-

drate, dansyl and mannose or mannose and galactose.

R

NH

NCH3H3C

SO

O

HN NH

O

S CO

HN

NHFe

HN

H3C H

CH3HO

HO

CH3

H

H

O

H

OHOHO

OH

O

HO

DNAOPO

OO

SNH

OR

DNAOPO

OO

AcSNH

OR

Thiol Click

OH

Figure 1. Structure of oligonucleotide conjugates synthesized

by "click thiol" chemistry

CONCLUSION

The TMTA is a very efficient click reaction to synthesize

oligonucleotides conjugated with various molecules. The

great advantages of this reaction are that it does not require

radical initiator and uv irradiation, and it occurs during the

deprotection and release of the oligonucleotide from solid

support affording the expected conjugates as the unique

molecule. TMTA could be applied to the synthesis of multi-

labelled oligonucleotides. Eventually, the sequential combi-

nation of the TMTA with the very popular CuAAC allowed

the synthesis of bis-conjugated oligonucleotides starting

either from the CuAAC and then TMTA or reverse.

REFERENCES 1. Singh, Y.; Murat, P.; Defrancq, E. Chem. Soc. Rev.

2010, 39, 2054-2070.

163

SYNTHESIS OF THE YPAA APTAMER BY CLICK LIGATION AND CONFORMATIONAL SWITCHING BY LIGAND SHAPE CONTROL

Maria Jenckel, Jennifer Frommer, Tamil Selvi Arunachalam, Bettina Appel and Sabine Müller*

Ernst Moritz Arndt Universität Greifswald, Institut für Biochemie, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany. *Correspondence to: smueller@uni-greifswald.de

ABSTRACT

We have investigated conformational switching in two

RNA systems: a small engineered hairpin ribozyme var-

iant and the ypaA aptamer of B. subtilis. Both RNAs are

responsive to flavine mononucleotide (FMN) and can be

conformationally controlled in dependence on the oxida-

tion state of FMN.

INTRODUCTION

Riboswitches play a very essential role in the regulation of gene expression. The FMN responsive riboswitch ypaA of B.

subtilis regulates expression of a gene encoding a riboflavin transport protein by effecting translation initiation. To study binding and folding of the ypaA aptamer domain, natural and specifically modified variants of the 129nts RNA were prepared in preparative scale using a combination of chemi-cal and enzymatic synthesis. A variety of methods was then used for binding and conformational analysis. Previously, we have developed a hairpin ribozyme variant responding to flavine mononucleotide (FMN) and several of its analogs [1-3]. FMN binds to the aptamer domain of the hairpin ribo-zyme derived aptazyme and stabilizes the catalytically com-petent conformation of the catalytic domain. Upon reduction, the molecular shape of FMN changes from planar to roof-like, leading to loss of its binding capacity, and subsequent-ly to down regulation of activity. Iterative cycles of oxida-tion/reduction allow for reversible switching of activity [2]. We have now used the established principle of FMN in-duced switching between two alternative RNA confor-mations with the ypaA aptamer.

RESULTS AND DISCUSSION

The aptamer domain of the ypaA riboswitch is a highly structured RNA, making its chemical preparation rather challenging. We started with the synthesis of aptamer frag-ments to be joined by enzymatic ligation. Screening several protocols, we succeeded with T4 RNA-Ligase splint ligation. Alternatively, ypaA aptamer variants were prepared by Click ligation using specifically functionalized RNA frag-ments (Figure 1). Folding of the ypaA variants was investi-gated in comparison to the natural aptamer by a number of methods. In particular, we used gelelectrophoretic gel mo-bility assays, CD and fluorescence spectroscopy and te-rahertz spectroscopy. Initial results indicate that the confor-mation of the ypaA riboswitch can be controlled by the oxi-

dation state of its ligand, analogous to the small engineered hairpin ribozyme variant described previously [1].

Figure 1. Click ligation used for generation of the ypaA ap-tamer.

Terahertz spectroscopy was carried out in collaboration

with the laboratory of Martina Havenith at Ruhr University Bochum, and showed a clear conformational transition of the RNA upon addition of FMN, associated with a change of water network dynamics. CD spectra of the aptamer rec-orded in the presence and absence of FMN as well as gel mobility analyses also clearly indicated FMN responsive conformational transitions. Furthermore, based on our pre-vious work on electrochemical reduction/oxidation of FMN, we have set up a new electrochemical assay involving im-mobilisation of the ligand on the electrode, to follow bind-ing and FMN dependent conformational switching.

CONCLUSION

Click-ligation was shown being an appropriate method for generation of functional ypaA aptamer variants. This allowed us to study FMN binding and conformational switching by a variety of methods, amongst them a novel electrochemical assay. The conformation of the aptamer can be controlled by changing the FMN oxidation state.

REFERENCES

1. Strohbach, D., Novak, N., Müller, S., Angew. Chem. Int.

Ed., 2006, 45, 2127-2129.

2. Strohbach, D., Turku, F., Schuhmann, W., Müller, S., Electroanalysis, 2008, 20, 935-940.

3. Müller, S., Appel, B., Krellenberg, T., Petkovic, S., IUBMB Life, 2012, 64, 34-47.

164

SYNTHESIS OF THE LIGHT-DRIVEN MOLECULAR MOTORS CONJUGATED WITH PEPTIDE

AND EVALUATION OF THE DNA BINDING PROPERTIES

Fumi Nagatsugi1,* Yusuke Takahashi

1, Maiko Kobayashi

1 and Shunsuke Kuwahara

2

1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai-shi, Miyagi, 980-8577, Japan.

2Graduate School of Science Toho University, 2-2-1 Miyama ,Funabashi-shi ,Chiba, 274-

8510 * Correspondence to: Email address for nagatugi@tagen.tohoku.ac.jp

ABSTRACT

Synthetic light-driven molecular motors are molecular

machine capable of rotation by photo-irradiation. In this

presentation, we wish to report the synthesis of the mo-

lecular motors conjugated with peptide and the evalua-

tion of the DNA binding properties.

INTRODUCTION

Synthetic molecular motors are one of an attractive mo-

lecular machine, which is capable of rotation under energy.

A variety of molecular motor systems have been designed in

past 10 years in which changes in shape, switching process-

es, or movements occur in response to external chemical,

electrochemical, or photochemical stimuli. But the applica-

tion of molecular motor in the movement of larger-scale

objects is limited and the useful functions of the molecular

motor have not been established. In this presentation, we

wish to report the development of the molecular motor,

which has the possibility to bind DNA duplex. DNA bind-

ing molecules have significant potential applications in ge-

nomic studies. Recently, the dynamic higher ordered DNA

structural change can play important roles in many cellular

processes. The small molecules inducing the structural

change of DNA have the potential to control the gene ex-

pression. We consider that the photo-driven molecular mo-

tor to bind DNA duplex has the potential to lead the struc-

tural change by photo irradiation. We have designed the

peptide-conjugated molecular motor, developed by Feringa 1and Harda

2, which can rotate in one rotational direction

through conversion of light energy into motor rotation

RESULTS AND DISCUSSION, CONCLUSION

The peptide conjugated molecular motors were synthesized

by using click chemistry between the molecular motor (1)

and two kinds of peptide derivatives (2) in good yields

(Scheme 1).

Trans-molecular motor (3a) and (4a) were rotated to cis-

olefin (3b) and (4b) by irradiated with UV light at 375 nm

for 15 s and heated at 37 ºC for overnight. Cis-molecular

motor (3b) and (4b) were also rotated to trans-olefin (3a)

and (3b) by irradiated with UV light at 415 nm for 15 s and

heated at 37 ºC for overnight. Binding affinity of the molec-

ular motors with duplex DNA was estimated by the ethidi-

um bromide displacement assay. Table 1 summarizes the

binding affinity of molecular motors (3) and (4) to duplex

DNA.

Peptide-conjugated trans-molecular motors (3a) and (4a)

showed lower C50 values toward all sequences of DNA than

those of cis-molecular motors (3b) and (4b). These results

have indicated that the binding affinity of trans-molecular

motor might be higher than that of cis-molecular motor.

In conclusion, we have successfully developed the molec-

ular motors to bind duplex DNA. We would like to present

these results in detail.

REFERENCES 1. ter Wiel, M. K. J.; van Delden, R. A.; Meetsma, A.; Feringa,

B. L., J. Am. Chem. Soc. 2003, 125, 15076-15086.

2. Fujita, T.; Kuwahara, S.; Harada, N., Eur. J. Org. Chem.,

2005, 4544-4556.

hn (312 nm)

hn (430 nm)

hn (330 nm)

D

hn (430 nm)

D

Fig.1. Light powered molecular motor

Scheme 1. Synthesis of the peptide conjugated molecular

motor

N3

N3

sodium ascorbate,

aq. CuSO4

DMF / H2O

N

N

N

N

N

N

(H2C)5

(CH2 )5

R

O

R

O

R (CH2)n

O

3a: trans R = KAKAK3b: cis R = KAKAK4a: trans R = PRGRP4b: cis R = PRGRP

1

2N

N

NN

N

N(CH2)3(H2C)3

RO

RO

hv (375 nm)and

hv (415 nm)and2a: R=KAKAK

2b: R=PRGRP

C50 (mM)3 (KAKAK) 4 (PRGEP)

PRGRP KAKAKtrans cis trans cis

DNA1 2.9 12.9 4.1 19.7 65.6 >100DNA2 2.3 5.6 1.7 10 >100 >100DNA3 3.1 4.9 2.7 8.2 >100 >100

1 mM DNA and 6 mM ethidium bromide in 5 mM cacodylate buffer containing 100 mM NaCl at pH 7.0

DNA3:5’CGCGCGCGCGCG3’

3’GCGCGCGCGCGC5’DNA1:5’CGCGAATTCGCG3’

3’GCGCTTAAGCGC5’

DNA2:5’CGATCGCGATCG3’

3’GCTAGCGCTAGC5’

Table 1. Binding affinity of molecular motors (C50, mM)

165

STUDY TOWARDS SYNTHESIS OF OLIGONUCLEOSIDES CONTAINING M1AA-DA, THE CONJUGATE MALONALDEHYDE-ACETALDEHYDE ADDUCT OF 2'-DEOXYADENOSINE

Donata Pluskota-Karwatka,* Kinga Salus and Henryk Koroniak

Adam Mickiewicz University, Faculty of Chemistry, Grunwaldzka 6, 60-780 Poznań, Poland. * Correspondence to: donatap@amu.edu.pl

ABSTRACT

The methodology for preparation of oligonucleosides

containing M1AA-dA, the conjugate malonaldehyde-

-acetaldehyde adduct of 2'-deoxyadenosine has been

investigated. The synthetic strategy includes assembly

of oligonucleotide from 2-deoxyadenosine modified

by the malonaldehyde-acetaldehyde conjugate. The key

features for preparation of this derivative include

synthesis of the adenine adduct and then

transribosylation by using nucleoside 2-

deoxyribotransferase.

INTRODUCTION, RESULTS AND DISCUSSION,

CONCLUSION

Endogenous, metabolic processes are sources of highly

reactive chemicals that are responsible for DNA damage.

Lipid peroxidation induced by so-called reactive oxygen

species (ROS), results in generating bifunctional

electrophiles, mainly carbonyl compounds and epoxides that

exhibit a wide range of biological activities. These

electrophiles can damage DNA by the reaction at electron

rich sites of nucleobases leading to the formation

of exocyclic adducts. Beside transformations caused directly

by ROS, exocyclic adducts are the most frequent reason

of DNA damage. For understanding of these biological

implications it is essential to synthesise oligonucleotides

containing the modified nucleosides placed in a structurally

specific manner.

One of the major carbonyl products of lipid peroxidation

is malonaldehyde. This aldehyde is known to form adducts

with the DNA bases. Two strategies have been reported for

preparation of oligonucleotides containing

the malonaldehyde adducts: assembly of oligonucleotides

from modified nucleosides and postoligomerisation

(postsynthetic modification strategy) [1, 2].

Our previous studies resulted in the identification

of a series of DNA adducts of malonaldehyde formed in the

presence of acetaldehyde [3]. These adducts consist of units

derived from malonaldehyde-acetaldehyde conjugates

and are formed in the reactions of DNA bases with

the appropriate malonaldehyde-acetaldehyde condensation

products (Scheme 1). The deoxyadenosine adduct M1AA-

dA, is one of the main products formed when calf thymus

DNA is allowed to react simultaneously with

malonaldehyde and acetaldehyde under physiological pH

and temperature [3].

This study deals with the developing of the strategy

leading to the synthesis of oligonucleotides containing

the propenoformyl adduct M1AA-dA.

O

O O

O

CH3

M1AA

M2AA

N

N

N

H3C

O

RO

N

N N

N

N

CH3

OO

dR

N

N N

N

N

dR

H3C

O

M1AA-dA M1AA-Cyd

N

N

N

CH3

O O

OR

N

N

N N

NO

O

CH3

dR

O

M2AA-dGuo I

HN

N N

N

N

O

O

H3C

O

dR

M2AA-dGuo II

M2AA-dA M2AA-Cyd

dG

O O

H3C H

dA, C

dA, C

O O

O O

H3C H

O+

Scheme 1. Formation of malonaldehyde-acetaldehyde

conjugate adducts

This study has been financially supported by National Centre

of Science, projects No N N204 433340.

REFERENCES

1. Schnetz-Boutaud, N. C., Mao, H., Stone, M. P., Marnett

L. J. Chem. Res. Toxicol. 2000, 13, 90-95.

2. Wang, H., Kozekov, I. D., Kozekova, A., Tamura, P. J.,

Marnett, L. J., Harris, T. M., Rizzo, C. J. Chem. Res.

Toxicol. 2006, 19, 1467-1474.

3. Pluskota-Karwatka, D., Pawłowicz, A. J., Kronberg, L.

Chem. Res. Toxicol. 2006, 19, 921-926.

166

SYNTHESIS AND PROPERTIES OF MODIFIED DNA OLIGONUCLEOTIDES WITH A

ZWITTERIONIC BACKBONE STRUCTURE

Boris Schmidtgall,1* Claudia Höbartner

2 and Christian Ducho

1

1University of Paderborn, Department of Chemistry, Warburger Str. 100, 33 098 Paderborn, Germany and

2MPI for

Biophysical Chemistry, Am Faßberg 11, 37 077 Göttingen, Germany. *Correspondence to: boris.schmidtgall@uni-paderborn.de

ABSTRACT

The site-specific replacement of the natural phospho-

diester linkage by the positively charged NAA (nucleosyl

amino acid) motif within oligonucleotides has been

performed, thus yielding DNA analogues with a

(partially) zwitterionic backbone structure. The

synthesis of such NAA-modified oligonucleotides and

studies on their properties are presented.

INTRODUCTION

Despite extensive studies on the structure and function of

nucleic acids there are still many open questions to consider.

Interestingly, it has been proposed that only uniformly

charged polymers are suitable for the formation of self-

sustaining systems [1,2]. However, there is only limited

experimental proof for this hypothesis. We have therefore

designed oligonucleotides (ON) with zwitterionic backbone

structures (Figure 1) which were inspired by the nucleoside

core motif of naturally occurring muraymycin antibiotics [3]

and termed as NAA (nucleosyl amino acid)-modified ON.

RESULTS AND DISCUSSION, CONCLUSION

For the solid phase-supported synthesis of ON bearing

the NAA-modification, dimeric phosphoramidite building

blocks (6'S)-1 and (6'R)-1 (Figure 2) were used. These

compounds were synthesised via peptide coupling of

3'-aminothymidine derivative 2 with diastereomerically pure

(6’S)- or (6’R)-nucleosyl amino acids (NAA) 3 and

subsequent manipulations of the protecting group pattern.

The highly diastereoselective synthesis of NAA structures

of type 3 has been developed and reported by us [4,5].

Using the phosphoramidite reagents (6’S)-1 and (6’R)-1,

several ON (20 to 22-mer) with mixed sequences were

synthesized with varying positions and numbers of

modifications within the backbone. Melting experiments

were perfomed to assess the thermal stability of DNA/DNA-

and DNA/RNA-duplices having one NAA-modified DNA

strand. Mismatch recognition of the NAA-modified ON was

also surveyed.

The melting point experiments indicated only a slight

decrease in thermal stability (ca. 0 to max. -2.2°C per NAA-

modification for DNA/DNA systems) and no significant

harmful influence on mismatch recognition. No structural

distortion of NAA-modified duplices was observed by CD-

spectroscopy. Consequently, our results do not support the

assumption of the uniform charge pattern to be necessary for

replicable biopolymers as genetic molecules and thus

challenge this hypothesis.

REFERENCES

1. Westheimer, F. Science, 1987, 235, 1173-1178.

2. Benner, S. A., Hutter, D. Bioorg. Chem., 2002, 30, 62-

80.

3. McDonald, L. A., Barbieri, L. R., Carter, G. T., Lenoy,

E., Lotvin, J., Petersen, P. J., Siegel, M. M., Singh, G.,

Williamson, R. T. J. Am. Chem. Soc., 2002, 124,

10260-10261.

4. Spork, A. P., Ducho, C. Org. Biomol. Chem., 2010, 8,

2323-2326.

5. Spork, A. P., Wiegmann, D., Granitzka, M., Stalke, D.,

Ducho, C. J. Org. Chem., 2011, 76, 10083-10098.

O

O

OB

O

O

BOP

OO

O

HN

OB

O

O

BO

H3N6'

* 5'5'

B = nucleobase

NAA-modified oligonucleotide

(6'S) or (6'R)

DNA

Figure 1. Native DNA and NAA-modified oligonucleotides.

Figure 2. Synthesis of dimeric phosphoramidites 1 for the stereocontrolled preparation of NAA-modified oligonucleotides.

167

BENZOPHENONE MODIFIED DNA FOR PHOTOCATALYSIS

Michael J. Weinberger and Hans-Achim Wagenknecht*

Karlsruhe Institute of Technology (KIT), Institute for Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany * Correspondence to: Weinberger@kit.edu

ABSTRACT

A Benzophenone – C – Nucleoside was synthesized

and incorporated into DNA to serve as a photosensitizer.

This modification allows the usage of light for chemical

reactions in structurally defined chiral vicinity.

INTRODUCTION

Using light and direct it in a certain way to a chemical

reaction via a photocatalyst combines demands of sustaina-

ble chemistry as well of modern catalysis. Moreover by add-

ing chiral information to the catalyst high enantioselectivi-

ties can be achieved. Benzophenone has already been ap-

plied successfully as a sensitizer for organocatalytic photo-

reactions by Bach and co-workers [1]. On the other hand

Feringa et al. [2] showed that DNA is a suitable source of

chiral information. We are now combining those two con-

cepts to build up DNA architectures with sensitizing abili-

ties. Therefore we synthesized the benzophenone C-

Nucleoside 1 and incorporated it into synthetic DNA. After

incorporation of 1, ss- and ds-DNA showed the benzophe-

none phosphorescence pattern [3]. Potentially we could use

this triplett energy to photocatalyse organic reactions, e.g.

[2+2]-cycloadditions or electron transfer induced radical

reactions.

RESULTS AND DISCUSSION

The key step during the synthetic route to 1 is the C-C-

coupling of a benzophenone derivative and Hoffer’s

Chlorosugar, which was performed in a Grignard reaction.

After deprotection to obtain 1 the following steps to the

DNA building block were standard phosphoramidite chem-

istry.

DNA modified with 1 clearly showed the benzophenone

phosphorescence pattern. These measurements gave the

evidence for the benzophenone triplet state in 1 and even in

DNA. To check for catalytic activity we chose the well-

known intramolecular [2+2]-cycloaddition of 4-(3-

butenyloxy)quinolone-2(1H)-one 2 [1,4-5].

The performed experiments using benzophenone or 1 in

- or -configuration as a catalyst worked efficient and

identical. This proofs that the modification of benzophenone

doesn’t affect the photophysical properties too much. But

before the modified Oligonucleotide itself can be used as an

efficient catalyst, it should also have the ability to bind the

substrate, thus to act as an aptamer.

To find a suitable aptamer sequence we are currently per-

forming SELEX on a random sequence 30-mer. We used

the quinolone derivative 5, where the carboxylic acid is

used as an anchor on oxirane modified polyacrylamide

beads.

After finding some aptamers targeting the quinolone

moiety, these sequences have to be modified by 1 and

checked for their ability to catalyse the benchmark reaction

of 2.

REFERENCES

1. Bauer, A., Wetkämper, F., Grimme, S., Bach, T. Nature,

2005, 436, 1139-1140.

2. Roelfes, G., Feringa, B. L. Angew. Chem. Int. Ed., 2005,

44, 3230-3232.

3. Weinberger, M., Wagenknecht, H.-A. Synthesis, 2012,

44, 648-652.

4. Kaneko, C., Suzuki, T., Sato, M., Naito, T. Chem.

Pharm. Bull., 1987, 35(1), 112-123.

5. Cauble, D., Lynch, V., Krische, M. JOC, 2003, 68, 15-

21.

168

COMPUTER MODELING OF THE CONFORMATIONAL PREFERENCES OF DAMAGED DNA

Stacey D. Wetmore*

Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive W, Lethbridge, Alberta, Canada *Correspondence to: stacey.wetmore@uleth.ca

ABSTRACT

Techniques in computational chemistry and models

ranging from damaged nucleobases to DNA helices pro-

vide an understanding of the conformational preferences

of C-linked deoxyguanosine phenolic adducts in duplex

environments. Through close collaborations with exper-

iments, conclusions can be drawn about the potential

biological effects of these lesions.

INTRODUCTION, RESULTS AND DISCUSSION,

CONCLUSION

The integrity of the genetic code is constantly threatened

by endogenous and exogenous sources that alter the chemi-

cal structure of DNA. Although many environmental con-

taminants have been identified as possible genotoxic agents

that covalently attach aryl groups to the DNA nucleobases,

little is known about the so-called bulky DNA “adducts”

(addition products). Furthermore, it appears to be difficult to

determine the exact mechanism of toxicity of these chemi-

cals, which is at least in part because of proposals that mul-

tiple mutations are induced when a single adduct adopts a

variety of conformations. In addition, the preferred confor-

mation of bulky adducts is dependent on a number of factors,

including adduct type, site of attachment, flanking bases,

complementary base, and environment (replication fork,

polymerase active site, etc.).

Without sufficient evidence that an environmental con-

taminant is a genotoxin, such as proof of a defined DNA

adduct structure, legislations that limit the extent of expo-

sure to humans via dietary intake, water, personal products,

etc., are significantly relaxed. Thus, studies of the exact

structure of DNA damage products and the possible muta-

genic mechanisms of compounds of potential risk are im-

portant for influencing legislative attitudes, as well as de-

veloping preventive and therapeutic measures for combating

potential carcinogenicity. As a result, we developed a sys-

tematic and graded computational approach to predict the

conformational and base-pairing preferences of the C-linked

phenolic 2-deoxyguanosine adducts (Figure 1). Specifically,

the nucleobase and nucleoside adducts are initially consid-

ered [1], and other factors are gradually incorporated into

the model including the backbone [2], complementary base

[3], flanking bases [4], and duplex environment [5].

Initial results from highly-accurate quantum chemical

calculations on small (nucleobase, nucleoside) models [1]

not only match the experimentally-predicted structure of the

adducts in a variety of solvents, but also explain observa-

tions regarding differences in orientations of the glycosidic

bond depending on the adduct formed. Although these stud-

ies predict a preference for the syn orientation about the

glycosidic bond, subsequent computational work considered

small models that are more relevant to B-DNA. For example,

calculations including the 5-monophosphate predict a very

small anti/syn energy difference [2], which suggests that a

complex conformational heterogeneity likely occurs in

DNA helices.

Using information gained by studying the preferred hy-

drogen-bonding patterns [3] and the effects of stacking with

the flanking nucleobases on the conformational preference

[4], the phenolic adducts were incoroprated into two strand

sequences, which differ in the nature of the bases flanking

the adduct. These sequences were studied both experimen-

tally and with molecular dynamics (MD) simulations [5].

Although experiments alone could not determine the con-

formation adopted by the adducts in DNA, the measured

melting temperatures combined with the calculated stabili-

ties of MD structures support a syn preference regardless of

the sequence or opposing base. This story highlights the

unique role computer calculations can play in elucidating

the structural preference of DNA damage when implement-

ed in conjunction with experiments, as well as unveiling the

biological implications of these lesions.

REFERENCES

1. Millen, A.L., Mclaughlin, C.K., Sun, K.M., Mander-

ville, R.A., Wetmore, S.D. J. Phys. Chem. A, 2008 112,

3742-3753 (2008).

2. Millen, A.L., Manderville, R.A., Wetmore, S.D. J. Phys.

Chem. B 2010, 114, 4373-4382.

3. Millen, A.L., Churchill, C.D.M., Manderville, R.A.,

Wetmore, S.D. J. Phys. Chem. B 2010, 114, 12995-

13004.

4. Millen, A.L., Kamenz, B.L., Leavens, F.M.V., Mander-

ville, R.A., Wetmore, S.D. J. Phys. Chem. B 2011, 115,

12993-13002.

5. Omumi, A., Millen, A.L., Wetmore, S.D., Manderville,

R.A. Chem. Res. Toxicol. 2011, 24, 1694-1709.

Figure 1. C-linked deoxyguanosine phenolic adducts.

169

COMPUTER MODELING OF DNA REPAIR ENZYMES: APPLICATIONS TO THE DNA GLYCOSYLASES

Stacey D. Wetmore*

Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive W, Lethbridge, Alberta, Canada *Correspondence to: stacey.wetmore@uleth.ca

ABSTRACT

A combined computational approach applied to DNA–

protein models can provide a greater understanding of

the mechanism of action of DNA repair enzymes. The

present contribution emphasizes the importance of such

applications by considering the DNA glycosylases, the

first enzymes involved in the base excision repair process.

INTRODUCTION, RESULTS AND DISCUSSION,

CONCLUSION

Although DNA can be damaged by a variety of sources,

intricate repair processes combat detrimental nucleobase

lesions. For example, the base excision repair (BER) pro-

cess uses several enzymes to identify and remove damaged

nucleobases from the DNA helix [1]. The first BER en-

zymes, the DNA glycosylases, are particularly intriguing

since they catalyse the cleavage of the inherently stable

base–sugar (glycosidic) bond, and some glycosylases are

bifunctional (exhibit -lyase activity).

Despite common themes in the proposed catalytic path-

ways for all glycosylases, conflicting conclusions and many

questions remain about the mechanism of events. Calcula-

tions can significantly contribute to this field since most

information has been conjectured from mutagenesis experi-

ments or crystal structures. The present contribution high-

lights recent work in the Wetmore group that combines

highly accurate quantum mechanical (QM) technqiues,

which describe reactions occuring in the active site, and

computationally efficient methodologies (MM, SE), which

treat the remainder of the DNA–enzyme complex.

Initial work investigated the effects of different factors

(nucleophile activation, substrate interactions) on the overall

barrier to glycosidic bond cleavage catalyzed by hUNG2 [2],

the glycosylase responsible for removing uracil (deaminated

cytosine) from DNA. A truncated active-site model (all res-

idues within approximately 10 Å of the substrate, Figure 1)

was developed for this most widely studied glycosylase,

which is justified by an abundance of reliable, high-

resolution crystal structures representing several steps of the

proposed reaction scheme. In this model, the substrate and

most important active-site residues were treated with DFT,

while the remaining atoms were treated with a lower-level

semi-empirical (SE) method. Using this ONIOM approach,

the relative energy was plotted as a function of the base–

sugar and nucleophile–sugar distances to identify the most

feasible reaction pathway. For the first time, a catalytic

pathway consistent with all current experimental data was

characterized. Furthermore, a modified mechanism was

proposed that explains a previously unresolved experimental

observation, and clarifies the identity and role of important

active site amino acid residues (including His268).

In contrast to hUNG2, the active site of the human en-

zyme responsible for repairing alkylation damage (AAG) is

lined solely with aromatic amino acids. Due to the low spec-

ificity of this enzyme and the lack of experimental crystal

structures that span the entire reaction and diversity of sub-

strates, the AAG catalytic mechanism was studied using a

computational model of the entire enzyme–DNA complex

(Figure 1) with neutral (ethanoadenine) and cationic (3-

methyladenine) lesions, as well as the natural A base [3].

The large size of this model requires an ONIOM approach

that uses DFT to treat the substrate and active site residues

and MM to treat the remainder of the system. This approach

accurately reproduces a recent experimental activation bar-

rier for the chemical step, and reveals important mechanistic

information including the (concerted) pathway used to ex-

cise both neutral and cationic lesions, and the dependence of

the role (catalytic bond cleavage or enhanced binding)

played by active-site aromatic amino acids on the lesion

being repaired. A novel explanation for why the natural A

base is not excised (poor nucleophilic activation) was also

developed, which suggests this enzyme may use the chemi-

cal step as a checkpoint to discriminate against natural pu-

rines.

REFERENCES 1. Berti, P. J., McCann, J. A. B. Chem. Rev. 2006, 106,

506-555.

2. Przybylski, J. L., Wetmore, S. D. Biochemistry, 2011,

50, 4218-4227.

3. Rutledge, L. R., Wetmore, S.D. J. Am. Chem. Soc. 2011,

133, 16258-16269.

Figure 1. ONIOM model for hUNG2 (left, DFT region in tube and SE region in wire frame) and AAG (right, DFT region dark blue and MM region in orange (mobile) and light green (fixed)).

170