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

该项目的目标是获得单链多核苷酸的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。在激光温度跳变后，将使用时间分辨率约为10纳秒的瞬态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巨大空白，即与单链崩溃和成核发夹形成相关的时间尺度和途径，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，无法提供完整的图像。特别是，这项工作将测试的假设，即在错误折叠构象的瞬态捕获是负责减少有效的扩散系数的链内动力学和所观察到的非阿耳修斯温度依赖性的关闭时间。将针对设计用于显著提高错配茎的概率的不同茎序列测量发夹形成的动力学;还将针对不同溶剂粘度测量动力学。 最近的一项统计力学研究的折叠/展开的21个核苷酸的RNA发夹，其中包括错误折叠状态的统计合奏，预测双相动力学与快速阶段发生在亚微秒。 这一预测将得到检验。 最后，单链两端之间的第一接触时间的直接测量将使用连接到链两端的供体和受体之间的三重态-三重态能量转移来测量。 为了验证理想链条预期的预测接触时间，将在链条的自相互作用最小化的条件下进行测量。 在增强自相互作用和诱导局部二级结构的溶剂条件下，将测量与发夹形成相关的接触时间。链多核苷酸将自身组织成简单的稳定结构，并将从这些简单结构中获得的见解扩展到对链内动力学和相互作用的更深入理解，更复杂的RNA结构。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地面测试，不仅是统计力学模型的细节，而且用于预测单链DNA和RNA的二级结构的热力学参数。主要教育目标是实施生物物理学本科课程。为吸引学生进入生物物理专业，将开发一门新的本科课程“生物物理学导论”。 两年前为高年级本科生和研究生开设了一门高级课程“分子和细胞生物物理学”，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.