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

职业生涯：使用实验校准的力场预测RNA折叠的高分辨率RNA三级结构核糖核酸(RNA)是一种多功能分子，在细胞内发挥着许多重要作用。小的microRNA可以结合和识别信使RNA，并控制它们翻译成蛋白质，而巨大的核糖核蛋白复合体可以完美精确地拼接基因。尽管RNA在现代细胞生物学中扮演着重要的角色，但目前预测RNA三维结构的方法是不够的，并且阻碍了我们识别RNA功能的潜在分子基础的能力。该项目需要开发改进的计算机模型，用于在3D原子分辨率下模拟RNA折叠。在这个项目中，我们发展了一种新的方法来校准RNA折叠力，其中利用RNA(核苷和核苷酸)在自然环境中构建块的实验性质来校准模型。改进后的模拟将被应用于解决RNA结构生物学中的两个棘手问题。第一个是确定3D结构如何在microRNA靶向中发挥作用，在这个过程中，数百个不同的信使RNA可以被单个microRNA靶向。第二个挑战是在解释RNA化学探测实验方面，对于3D结构未知的RNA来说，解释这些实验可能非常含糊。在这项研究计划的同时，还将实施一项教育计划，让那些来自弱势背景的学生接触STEM领域。为了做到这一点，化学专业的新生将一起生活，一起吃饭，一起参加每周一次的研讨会系列，以便让这些学生尽早接触到真正的科学职业(通过特邀演讲者)，并鼓励他们尽早参与本科研究。为了使分子动力学(MD)模拟能够准确地预测RNA的三级结构，原子学“力场”必须捕捉到单链区域的复杂行为，如环、凸起和螺旋连接。设计了一个热力学循环，用于根据实验确定的自由能来校准碱-碱相互作用的强度。通过这个过程，碱基堆积和碱基配对之间的溶剂化平衡可以针对每个碱基进行微调。最后，发展了一个协议，以二级结构轮廓为约束，通过分子动力学模拟确定RNA的三级结构。沙门氏菌Four-U RNA温度计将被用作一个模型系统，其中碱基对特定的熔化曲线可以直接与核磁共振进行比较。然后，该模型将被用于研究microRNA“密码”的结构基础，方法是测试膨胀动力学指示microRNA通过使用miR-34a(一种高度混杂的microRNA)识别mRNA靶标的假设。

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.