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. 2020 Dec 2;48(21):11982-11993.
doi: 10.1093/nar/gkaa999.

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

Marek Ondruš  1   2 , Veronika Sýkorová  1 , Lucie Bednárová  1 , Radek Pohl  1 , Michal Hocek  1   2
Affiliations

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

Marek Ondruš et al. Nucleic Acids Res. .

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.

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Figures

Scheme 1.
Scheme 1.
Design and synthesis of modified dNRTPs. Reagents and conditions: (i) R-C≡CH (excess), Pd(OAc)2 (7 mol.%), TPPTS (11 mol. %), CuI (8 mol.%), TEA (6 equiv.), AN/H2O 1:2, r.t., 16–48 h, under Ar, 69–98%; (ii) H2 (1 atm.), 10% Pd/C (10 mol. %), MeOH, r.t., 75°C, overnight, 93–98%; (iii) 1. POCl3 (1.2 equiv.), PO(OMe)3, -10°C, 2 h, under Ar; 2. (NHBu3)2H2P2O7 (5 equiv.), Bu3N (4 equiv.), DMF, -10°C, 1 h, under Ar; 3. 2 M TEAB; 18–28%; (iv) H2 (1 atm.), 10% Pd/C (10 mol. %), H2O, r.t., 4 h, 25%.
Figure 1.
Figure 1.
(A) Simultaneous incorporation of four modified dNRTPs using sequence specific templates in primer extension reaction. (B) Denaturing PAGE analysis of PEX reactions with set of modified dNERTPs and dNARTPs performed on 31-mer (lanes 1 and 7), 35-mer (lanes 2 and 8) 43-mer (lanes 3 and 9), 47-mer (lanes 4 and 10), 61-mer (lanes 5 and 11) and 98-mer (lanes 6 and 12) template using Vent (exo-) DNA polymerase. Lane L shows ssON ladder in corresponding sizes. (C) 31-mer ssONs achieved after PEX in various combinations of modified dNERTPs and dNARTPs followed by magnetoseparation. (D) Denaturing PAGE analysis of 31-mer PEX products in various combinations of dNERTPs and dNARTPs with negative (no dNTPs) and positive control (natural dNTPs) which serves as a size marker. Lanes are marked according to 1C.
Figure 2.
Figure 2.
Agarose gel of 5′-(6-FAM)-labelled PCR products using one modified dNRTP: modified dAEIn (lane 3), dAAIn (lane 4), dUEPh (lane 6), dUAPh (lane 7), dCEAlk (lane 9), dCAAlk (lane 10), dGEiPr (lane 12), dGAiPr (lane 13) or natural dNTPs (lane 1). Lanes 2, 5, 8 and 11 are control lanes with an absence of the particular modified dNRTP under study. PCR efficiencies were obtained relative to the PCR product with natural dNTPs (lane 1); (ds) double-stranded ladder. Fluorescence quantification was carried out using ImageJ.
Figure 3.
Figure 3.
The final combinatorial screen of modified dNRTPs in PCR. (A) Scheme of reverse and forward primers extension in PCR; (B) native agarose gel of PCR products with 5′-(6-FAM)-labelled reverse primer, Vent (exo-) DNA polymerase, 77-mer template and respective combination of two (lanes 1–5), three (lanes 6, 7) or four (lanes 8–12) modified dNRTPs; (C) cutout of native agarose gel of corresponding PCR products with Cy5-labelled reverse primer; (D) denaturing PAGE of extended 5′-(6-FAM)-labelled reverse primer in corresponding PCR products; (E) denaturing PAGE of extended Cy5-labelled forward primer in corresponding PCR products; (dsL) double-stranded ladder; (ssL) single-stranded ladder.
Figure 4.
Figure 4.
Native agarose gel of 5′-(6-FAM)-labelled aPCR products using four modified ethynyl-linked dNERTPs, Vent (exo-) and 77-nt (lane 3), 98-nt (lane 6), 120-nt (lane 9) and 150-nt (lane 12) templates. For each template, positive (lanes 1, 4, 7, 10, natural dNTPs) and negative controls (lanes 2, 5, 8, 11, no dNTPs) were carried out; (dsL) double-stranded ladder; (ssL) single-stranded ladder.
Scheme 2.
Scheme 2.
Asymmetric PCR synthesis using set of four modified dNERTPs, followed by re-PCR with natural dNTPs to prepare dsDNA for Sanger sequencing and NGS.
Figure 5.
Figure 5.
UV absorption spectra (A) and CD spectra (B) of 31DNA, 31DNA_NER and 31DNA_NAR; UV absorption spectra (C) and CD spectra (D) of sequence-heterogeneous 77DNA, 77DNA_NER and 77DNA_dsNERand their corresponding modified single strands (77ON_NER, 77cON_NER); all measurements were carried out in TrisHCl buffer (10 mM, 1 mM EDTA, 65 mM NaCl, pH 8.0).
Figure 6.
Figure 6.
CD spectra reflecting changes within natural 77DNA and modified 77DNA_NER, 77DNA_dsNER duplexes with increasing temperature (A-C), concentration of NaCl (D,F) and percentage of TFE (E).
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