Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al. 7, LT-10257 Vilnius, Lithuania.
Nucleic Acids Res. 2018 Jul 6;46(12):5911-5923. doi: 10.1093/nar/gky435.
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 N4-acylated 2'-deoxycytidine nucleotides (dCAcyl). We find that a variety of family A and B DNA polymerases efficiently use dCAcylTPs as substrates. In addition to the formation of complementary CAcyl•G pair, a strong base-pairing between N4-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 dCAcylTPs but is prone to form CAcyl•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N4-acylated-deoxycytidine nucleotides. These data reveal novel N4-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.
大量修饰的碱基被用作研究 DNA 和 RNA 的工具。由于实际原因，嘧啶的 C5 和嘌呤的 C7 是最适合修饰的位置。不幸的是，仅使用这两个位置，人们无法将修饰核苷酸的种类最大程度地扩展。在这里，我们展示了新型 N4-酰化 2'-脱氧胞苷核苷酸 (dCAcyl) 的合成和酶促掺入。我们发现各种 A 族和 B 族 DNA 聚合酶有效地将 dCAcylTPs 用作底物。除了形成互补的 CAcyl•G 对之外，当 Taq、Klenow 片段（外切）、Bsm 和 KOD XL DNA 聚合酶用于引物延伸反应时，N4-酰基胞嘧啶与腺嘌呤之间会发生强烈的碱基配对。相比之下，校对酶 phi29 DNA 聚合酶成功地利用了 dCAcylTPs，但在相同条件下容易形成 CAcyl•A 碱基对。此外，我们表明末端脱氧核苷酸转移酶能够掺入多达数百个 N4-酰化的脱氧胞苷核苷酸。这些数据揭示了新型 N4-酰化的脱氧胞苷核苷酸作为修饰 DNA 酶促合成的有益底物，可进一步应用于 DNA 片段的特定标记、适体的选择或光固定化。
