Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson–Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson–Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.

中文翻译：

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循环 RNA 3D 基序中非 Watson-Crick 碱基对的等排替换的上下文敏感性

研究了广泛的、反复出现的结构化 RNA 3D 基序 Sarcin/Ricin (S/R) 中的序列变异以解决三个相关问题：首先，由非 Watson-Crick 组成的结构化 RNA 3D 基序的稳定性如何（非 WC）碱基对，与相似长度和序列的 WC 配对螺旋相比？其次，对非 WC 对中等排和非等排碱基取代基序的稳定性有何影响？第三，在非 WC 碱基对中是否存在特定碱基组合的选择，这取决于生物体适应的温度状态？对适应不同温度的生物体的大小亚基 rRNA 的调查显示，在 S/R 基序的许多非 WC 配对位点存在系统性序列变异。含有该基序的寡核苷酸的紫外熔解分析和酶消化分析表明，更稳定的基序往往更坚硬。我们进一步发现，非 Watson-Crick 配对位点的碱基取代可以显着影响 S/R 基序的热力学稳定性，并且这些影响具有高度的上下文特异性，表明碱基堆叠和碱基-磷酸盐相互作用对基序稳定性的重要性。本研究强调了非规范碱基对的重要性及其对调节 RNA 分子稳定性和灵活性的贡献。我们进一步发现，非 Watson-Crick 配对位点的碱基取代可以显着影响 S/R 基序的热力学稳定性，并且这些影响具有高度的上下文特异性，表明碱基堆叠和碱基-磷酸盐相互作用对基序稳定性的重要性。本研究强调了非规范碱基对的重要性及其对调节 RNA 分子稳定性和灵活性的贡献。我们进一步发现，非 Watson-Crick 配对位点的碱基取代可以显着影响 S/R 基序的热力学稳定性，并且这些影响具有高度的上下文特异性，表明碱基堆叠和碱基-磷酸盐相互作用对基序稳定性的重要性。本研究强调了非规范碱基对的重要性及其对调节 RNA 分子稳定性和灵活性的贡献。