Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
J Biomol Struct Dyn. 2021 Apr;39(7):2555-2574. doi: 10.1080/07391102.2020.1751711. Epub 2020 Apr 16.
Sequence-specific ribonucleases are not found in nature. Absolute sequence selectivity in RNA cleavage normally requires multi-component complexes that recruit a guide RNA or DNA for target recognition and a protein-RNA assembly for catalytic functioning (e.g. RNAi molecular machinery, RNase H). Recently discovered peptidyl-oligonucleotide synthetic ribonucleases selectively knock down pathogenic RNAs by irreversible cleavage to offer unprecedented opportunities for control of disease-relevant RNA. Understanding how to increase their potency, selectivity and catalytic turnover will open the translational pathway to successful therapeutics. Yet, very little is known about how these chemical ribonucleases bind, cleave and leave their target. Rational design awaits this understanding in order to control therapy, particularly how to overcome the trade-off between sequence specificity and potency through catalytic turnover. We illuminate this here by characterizing the interactions of these chemical RNases with both complementary and non-complementary RNAs using T profiles, fluorescence, UV-visible and NMR spectroscopies. Crucially, the level of counter cations, which are tightly-controlled within cellular compartments, also controlled these interactions. The oligonucleotide component dominated interaction between conjugates and targets in the presence of physiological levels of counter cations (K), sufficient to prevent repulsion between the complementary nucleic acid strands to allow Watson-Crick hydrogen bonding. In contrast, the positively-charged catalytic peptide interacted poorly with target RNA, when counter cations similarly screened the negatively-charged sugar-phosphate RNA backbones. The peptide only became the key player, when counter cations were insufficient for charge screening; moreover, only under such non-physiological conditions did conjugates form strong complexes with RNAs.Communicated by Ramaswamy H. Sarma.
序列特异性核糖核酸酶在自然界中不存在。RNA 切割的绝对序列选择性通常需要多组分复合物，该复合物招募向导 RNA 或 DNA 以进行靶标识别，并组装蛋白-RNA 以进行催化作用（例如 RNAi 分子机制、RNase H）。最近发现的肽寡核苷酸合成核糖核酸酶通过不可逆切割选择性地敲低致病 RNA，为控制与疾病相关的 RNA 提供了前所未有的机会。了解如何提高其效力、选择性和催化周转率将为成功的治疗方法开辟转化途径。然而，人们对这些化学核糖核酸酶如何结合、切割和离开靶标知之甚少。为了控制治疗，特别是如何通过催化周转率来克服序列特异性和效力之间的权衡，合理的设计需要这种理解。通过使用 T 型谱、荧光、紫外可见和 NMR 光谱学来表征这些化学核糖核酸酶与互补和非互补 RNA 的相互作用，我们阐明了这一点。至关重要的是，细胞区室中严格控制的反离子水平也控制着这些相互作用。在生理浓度的反离子（K+）存在下，寡核苷酸成分主导着缀合物与靶标的相互作用，足以防止互补核酸链之间的排斥，从而允许 Watson-Crick 氢键形成。相比之下，当反离子同样屏蔽带负电荷的糖磷酸 RNA 骨架时，带正电荷的催化肽与靶 RNA 的相互作用很差。只有在反离子不足以为电荷屏蔽的情况下，肽才成为关键因素；此外，只有在这种非生理条件下，缀合物才会与 RNA 形成强复合物。由 Ramaswamy H. Sarma 传达。
