CAREER: Predicting high-resolution RNA tertiary structures using an experimentally callibrated force-field for RNA folding

职业：利用实验校准的RNA折叠力场预测高分辨率RNA三级结构核糖核酸（RNA）是一种多功能分子，在细胞内起着许多重要作用。小的microrna可以结合和识别信使RNA并控制其翻译成蛋白质，而巨大的核糖核蛋白复合物可以完美地拼接基因。尽管RNA在现代细胞生物学中发挥着重要作用，但目前预测RNA 3D结构的方法还不充分，并且阻碍了我们辨别RNA功能的潜在分子基础的能力。这个项目需要开发改进的计算机模型来模拟RNA折叠，在三维原子分辨率。在本项目中，开发了一种校准RNA折叠力的新方法，其中使用RNA构建块（核苷和核苷酸）在自然环境中的实验特性来校准模型。改进后的模拟将用于解决RNA结构生物学中的两个棘手问题。首先是确定3D结构如何在microRNA靶向中发挥作用，在microRNA靶向中，数百种不同的信使rna可以被单个microRNA靶向。第二个挑战是对RNA化学探测实验的解释，这对于3D结构未知的RNA的解释可能非常模糊。与该研究项目一起，将实施一项教育计划，让来自弱势背景的学生接触STEM领域。为了做到这一点，化学专业的大一新生将一起生活，一起吃饭，一起参加每周一次的系列研讨会，以便尽早让这些学生接触到真正的科学事业（通过邀请演讲者），并鼓励他们尽早参与本科生的研究。为了使分子动力学（MD）模拟能够准确预测RNA三级结构，原子“力场”必须捕捉单链区域的复杂行为，如环、凸起和螺旋结。设计了一个热力学循环，用于根据实验确定的自由能校准碱基相互作用的强度。通过这个过程，碱基堆叠和碱基配对之间的依赖溶剂的平衡可以为每个核碱基微调。最后，开发了一个协议，以确定RNA三级结构使用MD模拟使用二级结构概况作为约束。沙门氏菌四u RNA温度计将被用作模型系统，其中碱基对特定的熔化曲线可以直接与NMR进行比较。然后，该模型将用于研究microRNA“代码”的结构基础，通过测试凸起动力学通过使用miR-34a（一种高度混杂的microRNA）来决定microRNA对mRNA靶标的识别的假设。

CAREER: Predicting high-resolution RNA tertiary structures using an experimentally calibrated force-field for RNA folding Ribonucleic acid (RNA) is a versatile molecule that plays many important roles inside cells. Small microRNAs can bind to and recognize messenger RNA and control their translation into protein, while giant ribonucleoprotein complexes can splice genes with perfect precision. Despite RNA's important role in modern cellular biology, current methods for predicting RNA's 3D structure are inadequate and have hampered our ability to discern the underling molecular basis of RNA function. This project entails the development of improved computer models for simulating RNA folding, in 3D atomic resolution. In this project, a new approach to calibrate the forces for RNA folding is developed, in which experimental properties of building blocks of RNA (nucleosides and nucleotides) in their natural environment are used to calibrate the model. The improved simulations will be applied to tackle two vexing problems in RNA structural biology. The first is determining how 3D structure plays a role in microRNA targeting, in which hundreds of distinct messenger RNAs can be targeted by a single microRNA. The second challenge is in the interpretation of RNA chemical probing experiments, which can be highly ambiguous to interpret for RNAs whose 3D structure is unknown. Along with this research program, an education program will be implemented to expose those students coming from disadvantaged backgrounds to STEM fields. To do this, freshman chemistry majors will live, eat, and attend a weekly seminar series together in order to expose these students as early as possible to real scientific careers (through invited speakers) and encourage early participation in undergraduate research. In order for molecular dynamics (MD) simulations to accurately predict RNA tertiary structure the atomistic "force-field" must capture the complex behavior of single-stranded regions such as loops, bulges, and helical junctions. A thermodynamic cycle is devised for calibrating the strength of base-base interactions against experimentally determined free energies. Through this process, the solvation-dependent balance between base-stacking and base-pairing can be finely tuned for each nucleobase. Finally, a protocol is developed to determine RNA tertiary structures using MD simulations using secondary structure profiles as constraints. The salmonella four-U RNA thermometer will be used as a model-system where base-pair specific melting profiles can be directly compared with NMR. The model will then be used to investigate the structural basis of the microRNA "code", by testing the hypothesis that bulge dynamics dictates microRNA recognition of mRNA targets by using miR-34a, a highly promiscuous microRNA.