Bispecific circular aptamer tethering built-in universal molecular tag for functional protein deliveryXiaoshu Pan,a, ‡ Yu Yang,a, ‡ Long Li,a Xiaowei Li,a Qiang Li,a Cheng Cui,*a Bang Wang,a Hailan Kuai,b Jianhui Jiang,b Weihong Tan*a,b

a. Department of Chemistry and Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Health Cancer Center, UF Genetics Institute, McKnight Brain Institute, University of

Florida, Gainesville, Florida 32611-7200, United States.

b. Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, Aptamer

Engineering Center of Hunan Province, Hunan University, Changsha 410082, P. R. China.

S1

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2020

Table of Contents

Experimental Procedures…………………………………………………………………………………………………………………………………………………….S2

Results and Discussions ……………………………………………………………………………………………………………………………………………………….S4

Referecens…………………………………………………………………………………………………………………………………………………………………….…...S13

Author Contributions………………………………………………………………………………………………………………………………………………..….….…S13

S2

Experimental ProceduresReagents and materials. All materials used were purchased from commercial suppliers, unless stated otherwise. CPG and phosphoramidite for synthesizing all oligonucleotides were purchased from Glen Research LLC and ChemGenes Corp. All organic solvents for standard phosphoramidite synthesis were purchased from Glen Research LLC and Sigma-Aldrich. His-tagged RNase A was purchased from Sino Biological. DNA grade water used in all experiments was from Fisher Scientific. Molecular biology grade potassium phosphate dibasic solids were purchased from Sigma-Aldrich. T4 DNA ligase, DNA Exonuclease I and III were purchased from NEB Inc. Phenol/Chloroform/Isoamyl alcohol (25: 24: 1), stabilized and saturated with 100 mM Tris-EDTA to pH 8.0, were purchased from Acros Organics under Fisher Scientific. H-tagged Cas9 protein and H-tagged Cas9-GFP proteins were purchased from GeneScript.

Synthesis of oligonucleotides. All oligonucleotides used were synthesized with standard 1 µmol-scale phosphoramidite chemistry on an ABI 3400 DNA synthesizer (Applied Biosystmes Inc., Foster City, CA). 1000 Å controlled pore glass (CPG) was used to start. All oligonucleotides were deprotected from CPG according to the instructions from the manufacturer. The resulting oligonucleotides were purified by reversed-phase HPLC (ProStar, Varian). Then the collected oligonucleotides were vacuum-dried and further deprotected according to the manufacturer’s instructions. The ready-to-use oligonucleotides in DNA water were quantified by measuring the absorbance at 260 nm with a Cary Bio-300 UV Spectrometer (Varian). Details of sequences were listed in Table S1.

Ligation and purification of bc-apt. 3 µM anti-His tag aptamers were equivalently mixed with sgc8, a cell-specific aptamer in 1X T4 ligase buffer. The solutions were then heated at 95 °C for 8 mins and then cooled down completely on ice for 10 mins. 20-50 units of T4 ligase were used in 1000 µL of circular aptamer ligation. The ligation reaction was performed in a 1.5 mL Eppendorf tube in a shaker (Multi-Therm Shaker, temperature control, Alkali Scientific) at 16 °C with 800 rpm overnight (at least 16 hours). The reaction was then ended by heating at 75 °C for 10 min. Phenol/Chloroform/Isoamyl alcohol (25: 24: 1) was added into tubes with an equal volume to each ligation solution. The samples were vortexed for 5 min, left to sit for 5 min, and then centrifuged down at 120,000 rpm for 10 min. The supernatant was kept and then further purified by extraction two more times. The solutions were crudely purified and concentrated with 30K and 100K Ultrafiltration kit. 8% 6 M urea PAGE gel was made to separate pure bispecific circular aptamer (bc-apt) -- circular anti-His tag aptamer-sgc8 from side products. The gel was either illuminated by B-BOX Blue Illuminator or portable UV lamp (260nm), and bands representing the bc-apt were sliced off. The gel slices were transferred into a 5 mL syringe and submerged in the syringe with diffusion buffer 0.1 M TEAA (Triethylammonium Acetate). Diffusion buffer exchange was conducted every four hours, and then sample was separated by a 0.22 µm syringe filter from gel slices. The resulting bc-apt and monomers were then analysed by analytical anion exchange chromatography. The column DNA-Pac 4 x 250mm were used for analytical analysis of bc-apt, with 20mM NaOH as running buffer A, 1 M NaCl in 20 mM NaOH as elution buffer B with flow rate 1mL/min.

Stability of bc-apt. 1.5 µM bc-apt and linear bispecific aptamer (IDT Inc.) were incubated with 1 unit of Exonuclease I and III at 37 °C for 30 min and then heated at 75 °C for 10 min. 1.5 µM fluorescently labelled bc-apt and the mixture of 1.5 µM sgc8 and 1.5 µM anti-His tag aptamer were incubated with RPMI-1640 with 10% FBS (Fetal Bovine Serum, Gibco) from 0 hour to 36 hours. The treated solutions were then kept in a -20 freezer for later use in flow cytometry analysis and gel electrophoresis assay. The DNA in 3% agarose gel was stained by Ethidium Bromide and the bands were then quantified by ImageJ for further analysis.

Construction and characterization of binding between bc-apt and protein. H-tagged proteins, EGFP and RNase A, Cas9 proteins were used in the positive experimental group, while BSA (Bovine Serum Albumin) without His tag was used as control. 1 µM H-tagged EGFP, as target protein, and equal amount of BSA without His tag, as control nontarget, were incubated with 0.1 µM of bc-apt and 0.2 µM of anti-His tag aptamer in 50 mM K2HPO4 and 150 mM NaCl, anti-His tag aptamer binding buffer at 4 °C shaking at 800 rpm respectively.

S3

2 µM RNase A and comparable amount of BSA without His tag were individually incubated with 0.4 µM of linear anti-His tag aptamer and 0.2 µM bc-apt under the same condition described previously. Then, 24 pmol of protein, 2.4 pmol bc-apt, and 4.8 pmol anti-His tag aptamer were loaded into each well of 8-10% Native PAGE gel. Samples were run in 1X Tris-Glycine or 1X Tris-EDTA Borate native running buffer for 45-60 min at 110V or 150V. Then the gel was stained by Ethidium Bromide for 20 minutes and destained with ultrapure water for 10 min. Images were taken by Typhoon Imager. Next, the gel was stained by Coomassie blue for 30-60 min and destained with ultrapure water 4 times every 40 min, and images were also taken by Typhoon Imager.

2 µM H-tagged Cas9 proteins, as target protein, and equal amount of BSA without His tag, as control nontarget, were incubated with 0.2 µM of FAM labelled bc-apt in 50 mM K2HPO4 and 150 mM NaCl, anti-His tag aptamer binding buffer at 4 °C shaking at 800 rpm respectively. 8-10% Native PAGE gel. Samples were run in 1X Tris-Glycine native running buffer for 45min at 120V. Then the gel was stained by Gelred for 10min and then destained with ultrapure water for 20min. Images were taken by Typhoon Imager by Cy2 and Cy3 channel.

Binding assays. 1. Bc-apt/protein complex. 3 µM EGFP were first incubated with 15 µM bc-apt in 25 µL of 50 mM K2HPO4, 150 mM NaCl, 4.5 g/L glucose, 5 mM MgCl2, 50 µg/mL BSA, and 0.1 mg/mL yeast tRNA binding buffer at 4 °C for 2 hrs. Then, bc-apt/EGFP complex and free EGFP were incubated with 100K CCRF-CEM cells in 75 µL of the binding buffer mentioned above at 4 °C for another 30 min in a shaker. Cell pellets were centrifuged down at 300x g for 5 min, and supernatants were discarded. Next, cell pellets were washed with two volumes of 1x PBS (Dulbecco’s phosphate-buffered saline with Ca2+ and Mg2+, Sigma) and centrifuged down at the same condition. Suspended cell pellets in PBS and EGFP signal were collected through flow cytometry (BD Accuri C6) for the binding analysis. The experiments were conducted in triplicate. The data were analyzed by FlowJo.

0.8 µM Cas9-GFP were first incubated with 5 µM bc-apt in 50 µL of 50 mM K2HPO4, 150 mM NaCl, 4.5 g/L glucose, 5 mM MgCl2, 50 µg/mL BSA, and 0.1 mg/mL yeast tRNA binding buffer at 4 °C for 2 hrs. Then, bc-apt/Cas9-GFP complex and free Cas9-GFP were incubated with 75K CCRF-CEM cells in 50 µL of the binding buffer mentioned above at 4 °C for another 30 min in a shaker. Next, cell pellets were washed with two volumes of 1x PBS. Suspended cell pellets in PBS and EGFP signal were collected through flow cytometry (BD Accuri C6) for the binding analysis. The experiments were conducted in triplicate. The data were analyzed by FlowJo.

2. bc-apt pretreated CCRF-CEM cells. 2.5 µM bc-apt was used to pretreat 50K CCRF-CEM cells in binding buffer at 4 °C for 20 min. And then cells were washed once before adding 10 µM EGFP and then the binding buffer bringing to the equivalent volume. The EGFP and cells were incubated for 15 min at the same condition. And then use two-volume of cold PBS to remove non-specific binding. The one volume of PBS is used to resuspend CEM cells for flow cytometry analysis.

6 µM bc-apt was used to pretreat 50 CCRF-CEM cells in binding buffer at 4 °C for 20 min. And then cells were washed once before adding 15 µM EGFP and 15 µM EGFP with equiv. anti-His tag aptamer and sgc8, and then the binding buffer bringing to the equivalent volume. The EGFP and cells were incubated for 15 min at the same condition. And then use two-volume of cold PBS to remove non-specific binders. The one volume of PBS is used to resuspend CCRF-CEM cells for flow cytometry analysis.

3. FAM-bc-apt and mixture of FAM-sgc8 and anti-His tag aptamer. The aptamers treated with serum were three-fold diluted in the binding buffer and incubated with 100K cells at 4 °C for 30 min. Cell pellets were centrifuged down at 300x g for 5 min, and supernatants were discarded. Next, cell pellets were washed with two volumes of 1x PBS and centrifuged down at the same condition. Suspended cell pellets with PBS and FAM signal were collected through flow cytometry for binding analysis. The experiments were conducted twice.

Internalization assays. 350K CCRF-CEM and Ramos cells were washed with PBS and resuspended in Opti-MEM. 20 µM bc-apts were pretreated with the same amount of EGFP in the buffer containing 50 mM K2HPO4 and 150 mM NaCl and then incubated with 350K CCRF-CEM and Ramos cells with bc-apt/EGFP complex in the serum-free RPMI-1640 medium for 30min at 4 °C and then for 30min at 37 °C with 0.5µL/100 µL Lipofectamine 2000 in Opti-MEM medium.

S4

After centrifuge, the suspension was removed, and cold PBS was used to rinse CCRF-CEM and Ramos cells twice. Cells were resuspended in the serum-free RPMI-1640 medium before fluorescence imaging and let cells settle down for 5min in the confocal dish. The fluorescence images were consequently taken under a Leica TCS SP5 confocal microscope. The images were analyzed by LAS AF.

Cell-free bioactivity of RNase A. 480 µg tRNA transferred from baker's yeast, were incubated with the same amount of His-RNase A, bc-apt/His-RNase A, anti-His apt/His-RNase A, bc-apt and anti-His apt respectively in the binding buffer. Then each sample was incubated at 37°C for one hour and heated at 95°C for 5 min. 120 µg tRNA were loaded into 3% agarose gel for analysis.

Cell viability study. For intracellular delivery of His tag RNase A, CCRF-CEM cells and Ramos cells (30,000 cells) were incubated with 6 µM bc-apts in binding buffer at 4°C for 30 min and then seeded in a U-bottom 96-well plate. Unbound sequences were removed afterwards. 18 µM His tag RNase A in binding buffer were then added to cells and incubated for 30 min at 4°C. Supernatant was removed after incubation at 4°C for 30 min. Pretreated CCRF-CEM and Ramos cells per well in three aliquots and incubated in 100 µL fresh full culture medium for 48 hours. After washing cells with full culture medium once, MTS reagent (20 µL CellTiter reagent) diluted in fresh medium (100 µL) was added to each well at 37 °C for 2-3 hr incubation. The 96-well plate was scanned by a VersaMax tunable microplate reader (Molecular Devices, Inc., Sunnyvale, CA, USA), and absorbance at 490nm was recorded and normalized to the signals from untreated cells. Experiments were performed in triplicate.

S5

Results and Discussion

The oligonucleotides were investigated in this work were shown in Table S1. The extended sequences in red on the 5’-terminus were chemically modified with phosphate group. There are 13 nucleotides in the base pairing of sgc8 and anti-His tag aptamer for ligation. In the sequence of sgc8, fluorescein-dT was inserted into as a replacement for a dT residue for the binding analysis of bc-apt.

Table S1. Oligonucleotide sequences used in this study

Name Detailed sequence information

Anti-His-Phos apt 5’- Phos-TGA CTG ATT TAC GGC TAT GGG TGG TCT GGT TGG GAT TGG CCC CGG GAG CTG GC -3’

Anti-His apt 5’- TGA CTG ATT TAC GGC TAT GGG TGG TCT GGT TGG GAT TGG CCC CGG GAG CTG GC -3’

FAM-sgc8-Phos apt 5’- Phos- CGT AAA TCA GTC AGT CTA ACT GCT GCG CCG CCG GGA AAA TAC TGT ACG GTT(FAM) AGA C-3’

sgc8-Phos apt 5’- Phos- CGT AAA TCA GTC AGT CTA ACT GCT GCG CCG CCG GGA AAA TAC TGT ACG GTT AGA C-3’

Letters in red in sgc8 and anti-His aptamer are complementary sequences. Letter in green and purple is to form secondary of structure of each aptameric monomer predicted from IDT OligoAnalyzer Tool.

S6

Figure S1. Characterization of purified sgc8, anti-His tag aptamer, and bc-apt. Elution time for sgc8, anti-His tag aptamer and bc-apt are 15.0min, 13.0min, 14.5min respectively.

S7

Figure S2. Quantitative analysis of reduction in the band intensity of bc-apt and linear aptamer after the treatment of serum at 37°C for various hours, from 1 to 36 hours. The results were obtained by using the following equation: normalized reduction in band intensity=reduction in band intensity= (band intensity at 0 hour – band intensity at specific hour)/band intensity at 0 hour.

Figure S3. Electrophoretic mobility shift assay of H-tagged Cas9 and bc-apt. 8% Native PAGE gel was used to analyze the binding between 2 µM H-tagged Cas9 and 0.4 µM FAM labeled bc-apt. From left to the right, they are BSA only, BSA with FAM-bc-apt, H-tagged Cas9 only and H-tagged Cas9 with FAM-bc-apt respectively.

S8

Figure S4. Flow cytometry plots of EGFP-bound live CCRF-CEM cells. From left to right, images are CCRF-CEM cells treated with PBS only, preformed bc-apt/ EGFP (5:1) and EGFP only. The dot graph was analyzed by FlowJo.

S9

EGFP EGFP EGFP

Free EGFPCell only bc-apts/EGFP

Figure S5. Investigation of specific binding of EGFP mediated by bc-apt where bc-apt firstly bind with CCRF-CEM cells. (A) Flow cytometry analysis of bc-apt/EGFP complex binding with cells. CCRF-CEM cells were incubated with 2.5 µM bc-apt/ 10 µM EGFP. (B) Statistical analysis of EGFP bound CCRF-CEM cells after the incubation with 2.5 µM bc-apt/ 10 µM EGFP. The experiments were conducted in triplicate. ANOVA statistical analysis, with *** P<0.001. (C) Flow cytometry plots of EGFP-bound live CCRF-CEM cells. From left to right, images are CCRF-CEM cells treated with PBS only, bc-apt/ EGFP, and EGFP only. The dot graph was analyzed by FlowJo.

S10

102 103 104 105 106 107

Sample namebc-apt+EGFP + CCRF-CEM + His-apt

bc-apt+EGFP + CCRF-CEM + sgc8bc-apt+EGFP + CCRF-CEMEGFP + CCRF-CEMbc-apt + CCRF-CEMCCRF-CEM only

EGFP

Figure S6. Flow cytometry analysis of EGFP-bound live CCRF-CEM cells. CCRF-CEM cells were treated with different conditions from bottom to top, PBS only, 6 µM bc-apt only, EGFP only, bc-apt with EGFP, bc-apt with equiv. sgc8 monomer and EGFP, bc-apt and EGFP with equiv. His apt.

Sample namebc-apt + Cas9-GFP + CCRF-CEMCas9-GFP + CCRF-CEMCCRF-CEM

EGFP

Figure S7. Flow cytometry analysis of Cas9-GFP-bound live CCRF-CEM cells. CCRF-CEM cells were treated with different conditions from bottom to top, PBS only, Cas9-GFP, and bc-apt with Cas9-GFP.

S11

In order to investigate the cytotoxicity of RNase A mediated by bc-apt, we first investigated the biocompatibility of the bc-apt. The viability of CCRF-CEM cells treated with bc-apts were comparable to the control group without the treatment of bc-apt. The results demonstrated the excellent biocompatibility of bc-apt. Besides, we investigated that the therapeutic index of RNase A with different incubation strategy. In the study of Fig. S8B, we firstly incubated bc-apt with RNase A for 2 hours in 50 mM K2HPO4 and 150 mM NaCl and then preformed bc-apt/RNase A complex only incubated with cells in Opti-MEM for 4 hours at 37℃. It was found that the preformed bc-apt/His RNase A showed the lower cytotoxicity than RNase A only while not significantly lower than His-RNase A only. We reasoned that the His-RNase A can enter CCRF-CEM cells nonspecifically under the physiological condition.[1-2] With the incubation with bc-apt firstly and then His tagged RNase A at 4℃, targeted cells were killed more efficiently than preformed bc-apt/His RNase A (Fig. S8B and Fig. 5B). We then used Ramos cells as negative control to answer whether first incubation with bc-apt and then His tagged RNase A would affect the cell viability of nontargeted cells. There is no detectable cytotoxicity induced by RNase A in nontargeted cells.

Figure S8. (A) Cytotoxicity assay of different concentrations of bc-apts incubated with target CCRF-CEM cells. The experiments were conducted in triplicate. (B) Cytotoxicity assay of different concentrations of preformed bc-apts/His-RNase A complex and free RNase A. From the left to the right: Cell only; 19.2 µM His-RNase A; 8 µM bc-apts/19.2 µM His-RNase A.

S12

Figure S9. (A) Confocal image of intracellular delivery of H-tagged EGFP in Ramos cells with or without bc-apt. Scale bar: 25 µm. (B) Cytotoxicity assay of RNase A in the negative Ramos cells with or without bc-apt. The experiments were conducted in triplicate.

References

1. T. Chao, L. D. Lavis, R. T. Raines, Biochemistry, 2010, 49(50), 10666-10673.2. G. A. Ellis, M. J. Palte, R.T. Raines, J. Am. Chem. Soc. 2012, 134(8), 3631-3634.

Author Contributions

X. Pan and Dr. Y. Yang designed and performed all experiments, analyzed the experimental results. X. Pan wrote the manuscript. Prof. W. Tan supervised and led the discussion, analysis of the work in which all authors were involved.

S13