N (4)-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA
A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotid...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158702/ https://www.ncbi.nlm.nih.gov/pubmed/29846697 http://dx.doi.org/10.1093/nar/gky435 |
Sumario: | A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotides to a maximum. Here, we demonstrate the synthesis and enzymatic incorporation of novel N(4)-acylated 2′-deoxycytidine nucleotides (dC(Acyl)). We find that a variety of family A and B DNA polymerases efficiently use dC(Acyl)TPs as substrates. In addition to the formation of complementary C(Acyl)•G pair, a strong base-pairing between N(4)-acyl-cytosine and adenine takes place when Taq, Klenow fragment (exo–), Bsm and KOD XL DNA polymerases are used for the primer extension reactions. In contrast, a proofreading phi29 DNA polymerase successfully utilizes dC(Acyl)TPs but is prone to form C(Acyl)•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N(4)-acylated-deoxycytidine nucleotides. These data reveal novel N(4)-acylated deoxycytidine nucleotides as beneficial substrates for the enzymatic synthesis of modified DNA, which can be further applied for specific labelling of DNA fragments, selection of aptamers or photoimmobilization. |
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