Département de biochimie et médecine moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, H3C 3J7, Canada.
Nucleic Acids Res. 2019 Apr 23;47(7):3739-3751. doi: 10.1093/nar/gkz098.
Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.
研究功能 RNA 中结构元件的动态变化对于更好地理解其机制以及设计具有新型功能的 RNA 至关重要。此前，我们通过修饰关键的亲吻环相互作用（KLI）对 Varkud 卫星（VS）核酶进行了合理的工程研究，并将其特异性切换到非天然发夹底物。我们鉴定了具有替代 KLI（核糖体 RNA L88/L22 和人类免疫缺陷病毒-1 TAR/TAR*）的功能性 VS 核酶变体，但它们的切割活性降低了约 100 倍。在这里，我们研究了 KLI 的动力学，以将动态特性与功能相关联，并提高设计核酶的活性。我们使用温度复制交换分子动力学确定，VS 核酶中的天然 KLI 支持其封闭和活性状态的构象采样，而替代 KLI 则显示出更受限制的运动。基于体外选择，通过部分破坏 KLI 但增加构象采样，可以显著提高具有 TAR/TAR* KLI 的 VS 核酶变体的切割活性。我们提出了一个用于底物结合的机制模型，其中 KLI 动力学有助于形成活性位点。我们的模型支持 RNA 的模块化性质，其中亚结构域结构和动力学有助于定义与 RNA 功能相关的热力学和动力学。
