Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Laurentiev Avenue, 630090 Novosibirsk, Russia.
School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Nucleic Acids Res. 2020 Nov 4;48(19):10662-10679. doi: 10.1093/nar/gkaa780.
Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. 'Smart' supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-β anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an 'in-line' attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.
体内有效敲低致病 RNA 是小分子药物和生物药物都未能满足的迫切健康需求。“智能”催化剂超分子组装提供了对靶向 RNA 的精确识别和有效破坏，而这在自然界中尚未发现。本文通过化学工程方法设计了肽寡核苷酸核糖核酸酶，使其在与靶 RNA 杂交时形成和攻击凸起环区域。催化肽通过中心修饰的核苷酸（1 型）或 RNA 识别基序中的无碱基糖残基（2 型）掺入，揭示了在生物性能和核糖核酸酶活性的严格结构需求方面的显著差异。1 型缀合物均无催化活性，而所有 2 型缀合物均以序列特异性方式切割 RNA 靶标，通过多次切割，包括附近折叠处，5-nt 凸起环（BC5-α 和 BC5L-β 差向异构体）的切割率高达 90%。分子动力学模拟为肽对 RNA 切割位点的可及性提供了结构解释，采用了“直线”进攻构象进行催化。RNA 结合特异性和切割后解离的杂交测定和酶探测表明，如何平衡以允许催化反应的周转，从而使疾病相关 RNA 的多个拷贝失活和治疗效果成为可能。
