Structured RNAs such as ribozymes must fold into specific 3D structures to carry out their biological functions. While it is well-known that architectural features such as flexible junctions between helices help guide RNA tertiary folding, the mechanisms through which junctions influence folding remain poorly understood. We combine computational modeling with single molecule Förster resonance energy transfer (smFRET) and catalytic activity measurements to investigate the influence of junction design on the folding and function of the hairpin ribozyme. Coarse-grained simulations of a wide range of junction topologies indicate that differences in sterics and connectivity, independent of stacking, significantly affect tertiary folding and appear to largely explain previously observed variations in hairpin ribozyme stability. We further use our simulations to identify stabilizing modifications of non-optimal junction topologies, and experimentally validate that a three-way junction variant of the hairpin ribozyme can be stabilized by specific insertion of a short single-stranded linker. Combined, our multi-disciplinary study further reinforces that junction sterics and connectivity are important determinants of RNA folding, and demonstrates the potential of coarse-grained simulations as a tool for rationally tuning and optimizing RNA folding and function.

中文翻译：

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通过合理设计连接拓扑来调节RNA折叠和功能

结构化的RNA（例如核酶）必须折叠成特定的3D结构才能发挥其生物学功能。众所周知，螺旋之间的柔性连接等结构特征有助于引导RNA三级折叠，但对连接影响折叠的机制的了解仍然很少。我们将计算模型与单分子Förster共振能量转移（smFRET）和催化活性测量相结合，以研究连接设计对发夹状核酶的折叠和功能的影响。各种连接拓扑的粗粒度模拟表明，空间和连接性的差异（不依赖于堆叠）会显着影响第三级折叠，并且似乎在很大程度上解释了先前观察到的发夹状核酶稳定性的变化。我们进一步使用我们的模拟来识别非最佳连接拓扑的稳定修饰，并通过实验验证发夹核酶的三向连接变异体可以通过短插入单链接头的特异性插入而得以稳定。综合起来，我们的多学科研究进一步强调了连接空间和连通性是RNA折叠的重要决定因素，并证明了粗粒度模拟作为合理调整和优化RNA折叠和功能的工具的潜力。