COLLABORATIVE RESEARCH: Uncovering the Kinetic Mechanism of Base-Pairing and Stacking in RNA Folding

该研究项目将利用先进的荧光波动光谱技术，结合最先进的生物物理理论和建模，开发RNA二级结构形成过程中分子内碱基配对和碱基堆叠的基本反应速率模型。待开发的模型将预测任何给定序列的RNA二级结构形成的所有实验观察到的动力学特性，包括反应速率、反应机制以及反应中间体的结构和稳定性。该模型的发展将基于设计的RNA发夹的折叠和展开动力学的实验-理论比较。几个相互竞争的理论反应速率模型将用于预测发夹的温度依赖折叠动力学。这些反应的动力学随后将用FFS进行实验评估。这些实验将测量反应速率，并确定在从纳秒到数百毫秒的广泛时间尺度内发生的反应中间体。不同的反应速率模型定性和定量地预测不同的折叠动力学，包括不同的阿伦尼乌斯图、反应中间体和反应时间尺度。仅凭理论计算无法确定哪个反应速率模型是准确的。因此，对不同模式预测的实验比较将揭示哪个速率模式与实验观测最一致。一旦确定了最可能的模型，将对其进行细化，以考虑溶剂粘度、热容量、温度以及动力学和热力学参数的反离子依赖性。该模型将通过预测任意长度和序列的RNA发夹折叠动力学和实验验证来验证。形成RNA的更复杂的三级结构的建模将从这一点开始。这个项目更广泛的影响包括开发一个模型，可以帮助研究人员揭示任何给定RNA序列的生物学功能。RNA是构成生命系统的基本分子之一，在许多生物过程中起着至关重要的作用。这项研究还将有助于教育和培训新一代科学家，他们有能力将严格的定量实验和理论研究工具应用于重要的生物学问题。最后，基于这项研究的新的教学材料将在生物和物理科学的界面上为新兴课程开发。

This research project will use advanced fluorescence fluctuation spectroscopy (FFS) techniques combined with state-of-the-art biophysical theory and modeling to develop an elementary reaction rate model for intramolecular base-pairing and base-stacking in RNA secondary structure formation. The model to be developed will predict all experimentally observable kinetics properties of RNA secondary structure formation for any given sequence, including reaction rates, reaction mechanisms, and the structures and stabilities of the reaction intermediates. Development of the model will be based on experimental-theoretical comparisons of the folding and unfolding kinetics of designed RNA hairpins. Several competing theoretical reaction rate models will be used to predict the temperature dependent folding kinetics of the hairpins. The kinetics of these reactions will then be assessed experimentally using FFS. The experiments will measure reaction rates and identify reaction intermediates occurring over a broad range of time scales, from nanoseconds to hundreds of milliseconds. Different reaction rate models predict qualitatively and quantitatively different folding kinetics, including different Arrhenius plots, reaction intermediates, and reaction time scales. It is not possible to determine which reaction rate model is accurate based on theoretical calculations alone. Experimental comparisons to different model predictions will thus reveal which rate model is most consistent with experimental observations. Once the most likely model has been identified, it will be refined to account for the solvent viscosity, heat capacity, temperature, and counterion dependencies of the kinetic and thermodynamic parameters. The model will be validated by predicting the folding kinetics of RNA hairpins with arbitrary length and sequence and verifying the predictions experimentally. Modeling of more complicated tertiary structure forming RNA will proceed from that point.The broader impacts of this project include the development of a model that can aid researchers in uncovering the biological function of any given RNA sequence. RNA is one of the fundamental molecular building blocks of living systems and plays a crucial role in a host of biological processes. This research will also contribute to the education and training of a new generation of scientists capable of applying rigorous quantitative experimental and theoretical research tools to important biological problems. Finally, new pedagogical materials based on this research will be developed for an emerging curriculum at the interface of the biological and physical sciences.