Beloglazova Natalia G, Fabani Martin M, Polushin Nikolai N, Sil'nikov Vladimir V, Vlassov Valentin V, Bichenkova Elena V, Zenkova Marina A
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
J Nucleic Acids. 2011;2011:748632. doi: 10.4061/2011/748632. Epub 2011 Sep 25.
Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.
位点选择性人工核糖核酸酶（aRNases）的设计是RNA靶向领域最具挑战性的任务之一。在此，我们设计并研究了在催化部分含有多个咪唑残基的基于寡核苷酸的aRNases，并系统地改变了切割构建体的结构。我们证明，缀合物的核糖核酸酶活性受到催化部分中咪唑残基的数量、构建体的催化咪唑基团与寡核苷酸之间连接子的长度以及连接连接子结构和寡核苷酸的锚定基团类型的强烈影响。活性最高的aRNases的分子建模表明，切割构建体的优选取向强烈取决于锚定基团的结构和连接子的长度。脱氧胸苷锚定基团的加入显著降低了切割基团定位在切割位点附近的概率，这可能是由于与相邻核苷酸残基的堆积相互作用。总之，所得结果表明动力学因素在位点特异性RNA切割中起重要作用。具有最灵活和延伸的切割构建体的缀合物表现出非常高的切割活性，这可能为咪唑残基在可裂解磷酸二酯键附近正确定位提供了更好的机会。
