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Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases
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 synthe...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10681718/ https://www.ncbi.nlm.nih.gov/pubmed/37870471 http://dx.doi.org/10.1093/nar/gkad893 |
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author | Kuprikova, Natalia Ondruš, Marek Bednárová, Lucie Riopedre-Fernandez, Miguel Slavětínská, Lenka Poštová Sýkorová, Veronika Hocek, Michal |
author_facet | Kuprikova, Natalia Ondruš, Marek Bednárová, Lucie Riopedre-Fernandez, Miguel Slavětínská, Lenka Poštová Sýkorová, Veronika Hocek, Michal |
author_sort | Kuprikova, Natalia |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10681718 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-106817182023-10-23 Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases Kuprikova, Natalia Ondruš, Marek Bednárová, Lucie Riopedre-Fernandez, Miguel Slavětínská, Lenka Poštová Sýkorová, Veronika Hocek, Michal Nucleic Acids Res Chemical Biology and Nucleic Acid Chemistry 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. Oxford University Press 2023-10-23 /pmc/articles/PMC10681718/ /pubmed/37870471 http://dx.doi.org/10.1093/nar/gkad893 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Chemical Biology and Nucleic Acid Chemistry Kuprikova, Natalia Ondruš, Marek Bednárová, Lucie Riopedre-Fernandez, Miguel Slavětínská, Lenka Poštová Sýkorová, Veronika Hocek, Michal Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases |
title | Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases |
title_full | Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases |
title_fullStr | Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases |
title_full_unstemmed | Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases |
title_short | Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases |
title_sort | superanionic dna: enzymatic synthesis of hypermodified dna bearing four different anionic substituents at all four nucleobases |
topic | Chemical Biology and Nucleic Acid Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10681718/ https://www.ncbi.nlm.nih.gov/pubmed/37870471 http://dx.doi.org/10.1093/nar/gkad893 |
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