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.

• Klíčová slova
• DNA, enzymatic syntheses, nucleotides, polymerases,
• MeSH
• DNA-dependentní DNA-polymerasy * metabolismus   chemie   MeSH
• DNA * chemie   chemická syntéza   MeSH
• imidazoly * chemie   MeSH
• katalytická doména MeSH
• kyseliny karboxylové * chemie   MeSH
• polymerázová řetězová reakce MeSH
• puriny MeSH
• sulfhydrylové sloučeniny * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 7-deazapurine MeSH Prohlížeč
• DNA-dependentní DNA-polymerasy * MeSH
• DNA * MeSH
• imidazole MeSH Prohlížeč
• imidazoly * MeSH
• kyseliny karboxylové * MeSH
• puriny MeSH
• sulfhydrylové sloučeniny * 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
• deoxyribonukleotidy * chemie   chemická syntéza   MeSH
• DNA-dependentní DNA-polymerasy * metabolismus   MeSH
• DNA * chemie   biosyntéza   chemická syntéza   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kationty chemie   MeSH
• polymerázová řetězová reakce MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• deoxyribonukleotidy * MeSH
• DNA-dependentní DNA-polymerasy * MeSH
• DNA * MeSH
• kationty MeSH

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) derived from 5-substituted pyrimidines and 7-substituted 7-deazapurines bearing anionic substituents (carboxylate, sulfonate, phosphonate, and phosphate). The anion-linked dNTPs were used for enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase containing one, two, three, or four modified nucleotides. The polymerase was able to synthesize even long sequences of >100 modified nucleotides in a row by primer extension (PEX). We also successfully combined two anionic and two hydrophobic dNTPs bearing phenyl and indole moieties. In PCR, the combinations of one or two modified dNTPs gave exponential amplification, while most of the combinations of three or four modified dNTPs gave only linear amplification in asymmetric PCR. The hypermodified ONs were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies including hybridization, denaturation, CD spectroscopy and molecular modelling and dynamics suggest that the presence of anionic modifications in one strand decreases the stability of duplexes while still preserving the B-DNA conformation, whilst the DNA hypermodified in both strands adopts a different secondary structure.

• MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA * chemie   MeSH
• nukleotidy * chemie   MeSH
• pyrimidiny MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• DNA * MeSH
• nukleotidy * MeSH
• pyrimidiny MeSH

A set of 6 base-modified 2'-deoxyadenosine derivatives was incorporated to diverse DNA sequences by primer extension using Vent (exo-) polymerase and the influence of the modification on cleavage by diverse restriction endonucleases was studied. While 8-substituted (Br or methyl) adenine derivatives were well tolerated by the restriction enzymes and the corresponding sequences were cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence resulted in blocking of cleavage by some enzymes depending on the nature and size of the 7-substituent. All sequences with modifications outside of the recognition sequence were perfectly cleaved by all the restriction enzymes. The results are useful both for protection of some sequences from cleavage and for manipulation of functionalized DNA by restriction cleavage.

• MeSH
• deoxyadeninnukleotidy chemie   MeSH
• deoxyadenosiny chemie   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA chemie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• restrikční endonukleasy typu II metabolismus   MeSH
• štěpení DNA * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2'-deoxyadenosine MeSH Prohlížeč
• deoxyadeninnukleotidy MeSH
• deoxyadenosiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• restrikční endonukleasy typu II MeSH
• Tli polymerase MeSH Prohlížeč