Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups

Abstract A set of modified 2′-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

for 13 C NMR). 31 P chemical shifts were referenced to H3PO4 (0 ppm) as external reference. Coupling constants (J) are given in Hz. Complete assignment of all NMR signals was achieved by using a combination of H,H-COSY, H,C-HSQC, and H,C-HMBC experiments. Mass spectra were measured on LCQ classic (Thermo-Finnigan) spectrometer using ESI or Q-Tof Micro (Waters, ESI source, internal calibration with lockspray). Preparative HPLC separations were performed on a column packed with 10 μm C18 reversed phase (Phenomenex, Luna C18). High-resolution mass spectra were measured on a LTQ Orbitrap XL (Hermo Fischer Scientific) spectrometer using ESI ionization technique. Chemicals were of analytical grade.

Synthesis of modified nucleotides -5´-triphosphorylation
Scheme S3. Reaction scheme of phosphorylation reaction described by method C.
Method C: PO(OMe)3 (1 mL) was added through a septum to an argon-purged flask containing modified nucleosides dN ER or dN AR (1 equiv.) followed by dropwise addition of POCl3 (1.2 equiv.) at -10 °C (ice bath + NaCl) and the reaction mixture was stirred for 2 h at -10 °C (Scheme S3). Content of ice-cooled mixture containing solution of (NHBu3)2H2P2O7 (5 equiv.) and Bu3N (4 equiv.) in dry DMF (1 mL) was added dropwise and the reaction mixture and stirred for another 1 h at -10 °C. The reaction was quenched by addition of aqueous 2 M TEAB (triethylammonium bicarbonate) (5 mL). Solvents were evaporated under vacuum and co-distilled with water three times. The product was purified by HPLC on a C18 column with use of linear gradient from 0.1 M TEAB in H2O to 0.1 M TEAB in H2O/MeOH (1:1) as eluent. Conversion to sodium salt by ion exchange resin Dowex 50WX8 followed by freeze-drying from water gave solid product (Table S3).

2) Experimental section -biochemistry General remarks
All agarose and PAGE gels were analysed by fluorescence or phosphorous imaging using Typhoon FLA 9500 (GE Healthcare). The MALDI-TOF spectra of modified oligonucleotides were measured on UltrafleXtreme MALDI-TOF/TOF (Bruker) mass spectrometer with 1 kHz smartbeam II laser technology. The matrix consisted of 3hydroxypicolinic acid (HPA)/picolinic acid (PA)/ ammonium tartrate in ratio 9/1/1. The matrix (1 μL) was applied to the target (ground steel) and dried down at room temperature.

General procedure for ssDNA generation via magnetoseparation
In PEX reactions undergoing magnetoseparation, 5´-biotinylated template and nonlabelled prim248short primer was used (Table S4). In order to obtain sufficient amount of modified ssDNA for MALDI-TOF measurement, PEX reactions were five times scaled-up.        MALDI-TOF spectra of longer ONs (>50nt) were not carried. For these ONs, we performed Sanger and NGS sequencing (see section 2.11 and 2.12).

Sanger sequencing
In order to prepare dsDNA for Sanger sequencing, re-PCR of modified non-labelled 97ON_N ER was performed with L20 and Flank primers using reaction conditions as described in section 2.10. Resulting natural 97bp DNA was purified by Qiaquick PCR purification kit. dsDNA (50 ng/µL) was sent for Sanger sequencing using L20_Seq+ and Flank_Seq+ (5 µL, 5 µM, each) in order to improve sequencing results of such short DNA ( Figure S14, S15).

High-throughput Next-generation Sequencing (NGS)
NGS was performed using Nextera XT 2 Mid-Output cartridge and as an alignment of unique sequences with frequency in 833 and 1048 total analyzed reads ( Figure S17). Figure S17. Alignment of the most abundant (frequency over 0.2%) sequences obtained from NGS sequencing of PCR products generated from modified 97ON_N ER . First columns represent frequency of each unique sequence in 833 and 1048 reads, second column shows aligned sequences. Differences (point mutations) to original target sequence (in bold) are shown in red. Primer region is underlined.  Figure S18, S51).

3) Experimental section -UV-VIS absorption and CD spectroscopy
UV-absorption measurements were performed on Cary 100 Bio UV/VIS Spectrophotometer with temperature controller (Varian). The spectra were recorded in 1 mm rectangular quartz cell, in temperature range 25 °C -95 °C with temperature increment 1 °C / min under 260 nm detection and were obtained from three cycles (6 ramps in total). Tm values (in °C) were calculated using first negative derivative of intensity over temperature.
The circular dichroism (CD) measurements were performed on a Jasco-1500 spectropolarimeter equipped with Peltier thermostated holder PTC-517 (JASCO Inc. Easton, MD, USA). The spectra were recorded in temperature range 5 °C -95 °C with temperature increment 5 °C in spectral range from 200 nm to 400 nm in 1 mm rectangular quartz cell with following experimental setup: standard instrument sensitivity, 1 nm bandwidth, a scanning speed of 10 nm/min, a response time of 8 s and one accumulation.
The temperature of the sample was kept constant during each data accumulation and the same experimental setup was used for temperature increase and decrease. After baseline substraction the final data were recalculated on the concentration of nucleotides S44 and expressed as molar differential extinction ΔƐ (cm -1 mol -1 ). The melting temperatures were calculated using program Sigmaplot 12.5 (Systat software) when sigmoid fitting was applied.
In order to obtain sufficient amount of ethynyl-and alkyl-modified DNA to measure absorption, CD spectra and Tm values, synthesis of 31bp DNA (31DNA or 31DNA_N ER or 31DNA_N AR ) was scaled-up by 100 PEX reactions using prb4basII template with natural and modified dN ER TPs / dN AR TPs as described in section 2.3. PEX products were purified by Qiaquick nucleotide removal kit (Qiagen) and diluted to 2 µM final concentration by TrisHCl buffer (10 mM, 1 mM EDTA, 65 mM NaCl, pH 8.0) and set for measurement ( Figure 5A-B, S19).
In order to obtain ethynyl-modified 77DNA_N ER , aPCR reaction was scaled-up by 50 aPCR reactions using MO77 template and modified dN ER TPs as described in section 2.9.
aPCR product, 77ON_N ER , was annealed to its complementary sequence (30 pmol of MO77 for each 20 µL of aPCR reaction) directly in Vent (exo-) DNA polymerase buffer (Thermopol buffer). 77DNA_N ER was purified by Qiaquick PCR purification kit (Qiagen) using protocol provided by the manufacturer, dissolved in TrisHCl buffer (10 mM, 1 mM EDTA, 65 mM NaCl, pH 8.0) to 2 µM final concentration and set for Tm and CD measurements ( Figure 5C-D, S19). Natural 77DNA was prepeared using MO77 template with conditions describes for positive controls in section 2.9. Obtained DNA was purified by Qiaquick PCR purification kit (Qiagen) and dissolved in TrisHCl buffer (10 mM, 1 mM EDTA, 65 mM NaCl, pH 8.0) to 2 µM final concentration.