Department of Chemistry, University of Illinois Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Nucleic Acids Res. 2024 Aug 27;52(15):8702-8716. doi: 10.1093/nar/gkae639.
DNA and RNA nucleobase modifications are biologically relevant and valuable in fundamental biochemical and biophysical investigations of nucleic acids. However, directly introducing site-specific nucleobase modifications into long unprotected oligonucleotides is a substantial challenge. In this study, we used in vitro selection to identify DNAzymes that site-specifically N-alkylate the exocyclic nucleobase amines of particular cytidine, guanosine, and adenosine (C, G and A) nucleotides in DNA substrates, by reductive amination using a 5'-benzaldehyde oligonucleotide as the reaction partner. The new DNAzymes each require one or more of Mg2+, Mn2+, and Zn2+ as metal ion cofactors and have kobs from 0.04 to 0.3 h-1, with rate enhancement as high as ∼104 above the splinted background reaction. Several of the new DNAzymes are catalytically active when an RNA substrate is provided in place of DNA. Similarly, several new DNAzymes function when a small-molecule benzaldehyde compound replaces the 5'-benzaldehyde oligonucleotide. These findings expand the scope of DNAzyme catalysis to include nucleobase N-alkylation by reductive amination. Further development of this new class of DNAzymes is anticipated to facilitate practical covalent modification and labeling of DNA and RNA substrates.
DNA 和 RNA 核苷碱基修饰在核酸的基础生化和生物物理研究中具有重要的生物学意义和价值。然而，直接将特异性的核苷碱基修饰引入未经保护的长寡核苷酸中是一个巨大的挑战。在这项研究中，我们使用体外选择的方法，通过使用 5'-苯甲醛寡核苷酸作为反应伙伴的还原胺化作用，鉴定了能够特异性地将外切核苷碱基胺基 N-烷基化的 DNA 酶，这些碱基包括 DNA 底物中的胞嘧啶、鸟嘌呤和腺嘌呤（C、G 和 A）核苷酸。新的 DNA 酶每个都需要一个或多个 Mg2+、Mn2+和 Zn2+作为金属离子辅助因子，kobs 值从 0.04 到 0.3 h-1，比带有支架的背景反应高约 104 倍的速率增强。当提供 RNA 底物代替 DNA 时，几种新的 DNA 酶具有催化活性。同样，当用小分子苯甲醛化合物代替 5'-苯甲醛寡核苷酸时，几种新的 DNA 酶也能发挥作用。这些发现将 DNA 酶催化的范围扩展到包括通过还原胺化进行的核苷碱基 N-烷基化。预计这种新的 DNA 酶类的进一步发展将有助于 DNA 和 RNA 底物的实际共价修饰和标记。
