Search for the Structural Basis of Biomacromolecular Function and Activity

我的实验室在几个方面取得了进展。首先，我们开发了一种利用原子力显微镜研究溶液中RNA构象动力学的新算法和方法。简而言之，我们现在能够直接可视化溶液中的单个RNA构象，并确定单个RNA分子的结构；计算RNA在溶液中的总构象空间。RNA分子是高度动态和构象不均匀的。这一进展意义重大，因为它使表征异质构象的单个分子成为可能，例如溶液中的RNA，而不是均匀构象的分子集合。我们已经测试，基准和应用我们的新方法和方法，研究RNA的结构动力学和构象空间在一些重要的RNA分子在溶液中。这些RNA包括HIV包装信号RNA、Rev反应元件（RRE） RNA、带/不带tRNA配体的T-box核开关RNA、带/不带tRNA配体的钴胺核开关RNA、COVID-19的3‘和5’-UTR RNA和RNaseP RNA（包括全长和核心颗粒）。三份重要的手稿正在审查中或即将提交。其次，我们已经证明了使用RNA设备控制和调节小鼠EL4细胞中PD-1基因表达的可行性。PD-1基因是肿瘤免疫治疗的关键基因之一。因此，这个项目是潜在的翻译。基本的想法是使用外部可控的RNA设备，这些设备对配体结合有反应。我们特意选择了fda批准的配体。这些装置是利用CRISPR/ cas9技术在t细胞的染色体上设计的。基于去年取得的进展，现在我们已经建立了使用Western和qPCR方法量化不同配体浓度下PD-1表达的程序和方案。我们还获得了一些RNA器件在细胞中的动力学特性的信息。我们目前正在使用lentii -libraries进行高通量筛选，目的是确定在哺乳动物细胞中具有高效率和理想动力学特性的最佳RNA设备。此外，我们在存在和不存在配体的情况下结晶了一个RNA器件，从而为高分辨率结构测定打开了大门。值得注意的是，任何RNA装置的结构以前从未被确定过。RNA的高分辨率结构可能使我们更好地理解配体在原子水平上引发的构象变化，并激发更有效的RNA器件的新设计。最后，我们在使用高容量DNA模板附着珠改进PLOR技术方面取得了重大进展（Liu et al., Nat. 2015）。我们的目标是能够合成具有选择性标记或修饰残基放置在所需位置的千碱基长mRNA。改进的PLOR的应用之一可能是制造有选择性地标记修饰伪尿苷的mRNA，而不是目前辉瑞或Moderna在COVID-19疫苗中使用的统一标记mRNA。

My lab has made progress on several fronts. First, we have developed a novel algorithm and a method using AFM to study RNA conformational dynamics in solution. Briefly, we are now able to directly visualize individual RNA conformers in solution and determine the structures of individual RNA molecules; compute the total conformational space of RNA in solution. RNA molecules are highly dynamic and conformational-heterogeneous. This development is significant because it makes it possible to characterize individual molecules of heterogeneous conformations, such as RNA in solution, as opposed to an ensemble of molecules of homogeneous conformation. We have tested, bench-marked and applied our new approach and method in studying the RNA structural dynamics and conformational space in a number of important RNA molecules in solution. These include the HIV packaging signal RNA, Rev response element (RRE) RNA, the T-box riboswitch with/without tRNA ligand, cobalamine riboswitch RNA w/wo ligand, the 3' and 5'-UTR RNA of the COVID-19 and the RNaseP RNA (both the full-length and core particle). Three significant manuscripts are either under review or to be submitted. Second, we have demonstrated the feasibility of using RNA devices to control and regulate the PD-1 gene expression in mouse EL4 cells. The PD-1 gene is one of the critical genes for cancer immunotherapy. Thus this project is potentially translational. The basic idea is to use externally controllable RNA devices that are responsive to ligand bindings. We purposefully choose an FDA-approved ligand. The devices are engineered in a chromosome of T-cells using the CRISPR/Cas 9 technique. Built on the progress in the last year, now we have established the procedure and protocol to quantify the PD-1 expression at various ligand concentrations using both Western and qPCR methods. We have also obtained information on the kinetic characteristics of some of the RNA devices in cell. We are currently performing high-throughput screenings using lenti-libraries with the aim to identify the best RNA devices that are both of high efficiency and ideal kinetic characteristics in mammalian cells. Furthermore, we have crystallized one of the RNA devices in both presence and absence of ligand and thus opened the door for high-resolution structure determination. It is noteworthy to mention that the structure of any RNA devices has never been determined before. The high-resolution structure of an RNA may lead us to a better understanding of the ligand-triggered conformation changes at the atomic level and stimulate new designs of more efficient RNA devices. Lastly, we have made significant progress in improving the PLOR technology (Liu et al., Nat. 2015) using high-capacity DNA template attachedbeads. Our aim is to be able to synthesize kilo-base long mRNA with selectively-labeled or modified residues placed at desired positions. One of the applications of the improved PLOR could be manufacturing mRNA selectively labeled with modified pseudo-uridines, as opposed to the current uniform labelings such as mRNAs in the COVID-19 vaccines by Pfizer or Moderna.