We designed and synthesized a set of six 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing functional groups mimicking amino acid side chains in enzyme active sites (OH, SH, COOH, and imidazole) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through different linkers. These modified dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using several DNA polymerases. In primer extension (PEX), all modified dNTPs provided DNA containing one, two, three, or, (all) four modified nucleotides each bearing a different modification, although the thiol-modified dNTPs were worse substrates compared to the others. In PCR, we observed exponential amplification for any combination of one, two, or three nonsulfur dNTPs but the thiol-modified dNTP did not work well in any combinations. Sequencing of the hypermodified DNA confirmed the good fidelity of the incorporation of all the modified nucleotides. This set of modified dNTPs extends the portfolio of building blocks for prospective use in selections of functional nucleic acids.

• Keywords
• DNA, enzymatic syntheses, nucleotides, polymerases,
• MeSH
• DNA-Directed DNA Polymerase * metabolism   chemistry   MeSH
• DNA * chemistry   chemical synthesis   MeSH
• Imidazoles * chemistry   MeSH
• Catalytic Domain MeSH
• Carboxylic Acids * chemistry   MeSH
• Polymerase Chain Reaction MeSH
• Sulfhydryl Compounds * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 7-deazapurine MeSH Browser
• DNA-Directed DNA Polymerase * MeSH
• DNA * MeSH
• imidazole MeSH Browser
• Imidazoles * MeSH
• Carboxylic Acids * MeSH
• Purines MeSH
• Sulfhydryl Compounds * MeSH

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing cationic substituents (protonated amino, methylamino, dimethylamino and trimethylammonium groups) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through hex-1-ynyl or propargyl linker. These cationic dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase. In primer extension (PEX), we successfully obtained DNA containing one, two, three, or (all) four modified nucleotides, each bearing a different cationic modification. The cationic dNTPs were somewhat worse substrates compared to previously studied dNTPs bearing hydrophobic or anionic modifications, but the polymerase was still able to synthesize sequences up to 73 modified nucleotides. We also successfully combined one cationic modification with one anionic and two hydrophobic modifications in PEX. In polymerase chain reaction (PCR), we observed exponential amplification only in the case of one cationic modification, while the combination of more cationic nucleotides gave either very low amplification or no PCR product. The hypermodified oligonucleotides prepared by PEX were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies of hybridization, denaturation, and circular dichroism spectroscopy showed that the presence of cationic modifications increases the stability of duplexes.

• MeSH
• Deoxyribonucleotides * chemistry   chemical synthesis   MeSH
• DNA-Directed DNA Polymerase * metabolism   MeSH
• DNA * chemistry   biosynthesis   chemical synthesis   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Cations chemistry   MeSH
• Polymerase Chain Reaction MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Deoxyribonucleotides * MeSH
• DNA-Directed DNA Polymerase * MeSH
• DNA * MeSH
• Cations MeSH

DNA modifications on pyrimidine nucleobases play diverse roles in biology such as protection of bacteriophage DNA from enzymatic cleavage, however, their role in the regulation of transcription is underexplored. We have designed and synthesized a series of uracil 2'-deoxyribonucleosides and 5'-O-triphosphates (dNTPs) bearing diverse modifications at position 5 of nucleobase, including natural nucleotides occurring in bacteriophages, α-putrescinylthymine, α-glutaminylthymine, 5-dihydroxypentyluracil, and methylated or non-methylated 5-aminomethyluracil, and non-natural 5-sulfanylmethyl- and 5-cyanomethyluracil. The dNTPs bearing basic substituents were moderate to poor substrates for DNA polymerases, but still useful in primer extension synthesis of modified DNA. Together with previously reported epigenetic pyrimidine nucleotides, they were used for the synthesis of diverse DNA templates containing a T7 promoter modified in the sense, antisense or in both strands. A systematic study of the in vitro transcription with T7 RNA polymerase showed a moderate positive effect of most of the uracil modifications in the non-template strand and some either positive or negative influence of modifications in the template strand. The most interesting modification was the non-natural 5-cyanomethyluracil which showed significant positive effect in transcription.

• Publication type
• Journal Article MeSH