Department of Biochemistry, School of Medicine, Stanford University, Stanford, CA 94305.
Department of Chemical Engineering, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2021 Aug 17;118(33). doi: 10.1073/pnas.2109085118.
Despite RNA's diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural "motifs"-helices, junctions, and tertiary contact modules-to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used "RNA on a massively parallel array" (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ∼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.
尽管 RNA 具有多样的二级和三级结构以及复杂的构象变化，但自然界利用有限的结构“基序”-螺旋、连接和三级接触模块-来构建多样的功能性 RNA。因此，深入描述相对较小的 RNA 基序宇宙可能会导致 RNA 三级构象景观的预测模型。基序的性质可能取决于序列和二级结构，从而产生扩展 RNA 构建块宇宙的子类。然而，鉴于在高通量中映射构象性质的挑战，我们对基序子类知之甚少。以前，我们使用“大量平行阵列上的 RNA”（RNA-MaP），一种定量的高通量技术，研究了数千个螺旋和双向连接。在这里，我们将 RNA-MaP 改编为研究四螺旋环/四螺旋环受体（TL/TLR）三级接触基序的热力学和构象性质，分析了来自不同类别的 1493 个 TLR 序列。聚类分析显示了 TL 特异性、稳定性和构象行为的可变性。尽管如此，天然的 GAAA/11ntR TL/TLR 在三级稳定性上变化约 2.5 kcal/mol，但表现出保守的 TL 特异性和构象性质。因此，RNA 可以在不改变这些 TL/TLR 整体结构的情况下调整稳定性。此外，它们的稳定性与自然频率相关，表明热力学是主要的选择压力。相比之下，其他 TL/TLR 显示出异质的构象行为，似乎不受强烈的热力学选择的影响。我们的结果构建了一个基于孤立基序性质的 RNA 折叠热力学的可推广模型，并且我们的表征的 TL/TLR 文库可用于设计具有可预测热力学和构象行为的 RNA。
