Time-Resolved Measurements of Secondary Structure Formation in Single-Stranded Polynucleotides

这个项目的目标是获得关于单链多核苷酸链内动力学的定量信息，并确定这些动力学如何影响二级结构形成的动力学。在激光温度跳跃后，将使用时间分辨率约为10纳秒的瞬时吸光度测量来监测动力学。这些实验旨在填补RNA分子折叠研究中的一个很大空白，即与单链链破裂和成核发夹形成相关的时间尺度和路径，这些时间尺度发生在几纳秒到微秒的时间尺度上。以前对发夹形成的测量仅限于微秒级的时间分辨率，未能提供完整的图像。特别是，这项工作将检验这一假设，即错误折叠构象中的瞬时陷阱负责降低链内动力学的有效扩散系数，并对观察到的闭合时间的非阿累尼乌斯温度依赖性负责。将对不同的茎序列测量发夹形成的动力学，以显著增加不匹配的茎的可能性；还将测量不同溶剂粘度的动力学。最近对21个核苷酸的RNA发夹的折叠/展开的统计力学研究，其中包括统计系综中的错误折叠状态，预测了两相动力学，快速相发生在亚微秒级。这一预测将得到验证。最后，直接测量单链链两端之间的第一次接触时间将使用连接到链两端的施主和受体之间的三重-三重态能量转移来测量。为了验证理想链的预计接触时间，测量将在链的自相互作用最小化的条件下进行。与发夹形成相关的接触时间将在增强自身相互作用并诱导局部二级结构的溶剂条件下进行测量。本研究的意义在于定量了解单链多核苷酸如何将自身组织成简单稳定的结构，并将从这些简单结构中获得的见解扩展到更深入地理解导致更复杂的RNA结构的链内动力学和相互作用。RNA折叠的动力学测量和统计力学研究之间的密切比较将提供一个理想的基础来测试，不仅是统计力学模型的细节，而且是用于预测单链DNA和RNA二级结构的热力学参数。主要的教育目标是实施生物物理学的本科课程。作为吸引学生进入生物物理学专业的一种方式，将为本科生开发一门新的课程--生物物理学概论。为高级本科生和研究生开设的高级课程“分子与细胞生物物理学”是两年前开发的，现在每年都开设。

The goals of this project are to obtain quantitative information on the intrachain dynamics of single-stranded polynucleotides, and to determine how these dynamics influence the kinetics of secondary structure formation. The kinetics will be monitored using transient absorbance measurements with ~ 10 nanosecond time-resolution after a laser temperature-jump. The experiments are designed to fill a big gap in the folding studies of RNA molecules, namely the time-scales and pathways associated with the collapse of a single-stranded chain and the formation of nucleating hairpins, which occur on time-scales of several nanoseconds-to-microseconds. Previous measurements on hairpin formation have been limited to microsecond time-resolution and have failed to provide a complete picture. In particular, this work will test the hypothesis that transient trapping in misfolded conformations is responsible for reducing the effective diffusion coefficient for intrachain dynamics and for the observed non-Arrhenius temperature dependence of the closing times. Kinetics of hairpin formation will be measured for varying stem sequences designed to significantly enhance the probability of mis-matched stems; the kinetics will also be measured for varying solvent viscosity. A recent statistical mechanical study of the folding/unfolding of a 21-nucleotide RNA hairpin, which included misfolded states in the statistical ensemble, predicted biphasic kinetics with a rapid phase occurring on sub-microseconds. This prediction will be tested. Finally, direct measurements of the first contact time between the two ends of a single-stranded chain will be measured using triplet-triplet energy transfer between a donor and an acceptor attached to the two ends of the chain. To verify the predicted contact times expected for an ideal chain, the measurements will be done under conditions where the self-interactions of the chain are minimized. The contact times relevant for hairpin formation will be measured under solvent conditions that enhance self-interactions and induce local secondary structure.The significance of this study is to develop a quantitative understanding of how a single-stranded polynucleotide organizes itself into simple stable structures and to extend the insights gained from these simple structures to a deeper understanding of the intrachain dynamics and interactions that lead to successively more complex RNA structures. Close comparison between kinetics measurements and statistical mechanical studies of the folding of RNA will provide an ideal ground to test, not only the details of the statistical mechanical models, but also the thermodynamic parameters used for predicting secondary structures in single-stranded DNA and RNA. The primary educational goals are to implement an undergraduate program in Biophysics. A new course for undergraduates "Introduction to Biophysics" will be developed as a way of attracting students into the Biophysics major. A higher level course "Molecule and Cell Biophysics" intended for advanced undergraduates and beginning graduate students was developed two years ago and is now offered each year.