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

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 dN...

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Autores principales: Ondruš, Marek, Sýkorová, Veronika, Bednárová, Lucie, Pohl, Radek, Hocek, Michal
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708046/
https://www.ncbi.nlm.nih.gov/pubmed/33152081
http://dx.doi.org/10.1093/nar/gkaa999
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author Ondruš, Marek
Sýkorová, Veronika
Bednárová, Lucie
Pohl, Radek
Hocek, Michal
author_facet Ondruš, Marek
Sýkorová, Veronika
Bednárová, Lucie
Pohl, Radek
Hocek, Michal
author_sort Ondruš, Marek
collection PubMed
description 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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spelling pubmed-77080462020-12-07 Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups Ondruš, Marek Sýkorová, Veronika Bednárová, Lucie Pohl, Radek Hocek, Michal Nucleic Acids Res Chemical Biology and Nucleic Acid Chemistry 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. Oxford University Press 2020-11-05 /pmc/articles/PMC7708046/ /pubmed/33152081 http://dx.doi.org/10.1093/nar/gkaa999 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://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
Ondruš, Marek
Sýkorová, Veronika
Bednárová, Lucie
Pohl, Radek
Hocek, Michal
Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups
title Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups
title_full Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups
title_fullStr Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups
title_full_unstemmed Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups
title_short Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups
title_sort enzymatic synthesis of hypermodified dna polymers for sequence-specific display of four different hydrophobic groups
topic Chemical Biology and Nucleic Acid Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708046/
https://www.ncbi.nlm.nih.gov/pubmed/33152081
http://dx.doi.org/10.1093/nar/gkaa999
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