Paul Somdeb, Wong Antonio A W L, Liu Leo T, Perrin David M
Chemistry Department, University of British Columbia, 2036 Main Mall, UBC, Vancouver, BC V6T-1Z1, Canada.
Chembiochem. 2022 Mar 4;23(5):e202100600. doi: 10.1002/cbic.202100600. Epub 2022 Jan 18.
Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dU TP) and 5-aminoallyl-2'-dCTP (dC TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.
在不存在二价金属阳离子（M）的情况下，核酸催化剂对RNA进行序列特异性切割一直是生物模拟领域的一个重要目标，具有潜在的治疗应用价值。鉴于天然核苷酸中官能团多样性的缺乏，带有类似氨基酸侧链的修饰核苷酸被用于提高单个嵌入核糖核苷位点的自我切割速率。先前的研究依赖于三个官能团：一个胺基、一个胍基和一个咪唑基，分别存在于三种不同的核苷上。然而，迄今为止，很少有研究系统地探讨这三种修饰是否都是必需的，因为任何单个修饰核苷的价值在选择之初就已被考虑在内。在此，我们报道仅使用两种修饰的dNTP（不包括咪唑），即5-(3-胍基烯丙基)-2'-dUTP（dUTP）和5-氨基烯丙基-2'-dCTP（dCTP），来筛选在不存在M的情况下以不依赖pH的方式进行切割的体外自我切割DNA酶。切割表现出双相动力学，其速率常数显著高于未修饰的DNA酶，并且与某些含有咪唑的DNA酶相比具有优势。
