Search for the Structural Basis of Biomacromolecular Function and Activity

寻找生物大分子功能和活性的结构基础

基本信息

批准号：
10926008
负责人：
Yun Xing m wang
金额：
$ 221.52万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10926008
关键词：
Algorithms Atomic Force Microscopy Benchmarking Biochemical Biological Biophysics COVID-19 COVID-19 vaccine Cells Characteristics Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Crystallization DNA Development Devices Engineering Event FDA approved Gene Expression Genes Goals HIV Individual Kinetics Knowledge Label Length Libraries Ligands Mammalian Cell Manuscripts Messenger RNA Methods Molecular Conformation Mus NMR Spectroscopy Nucleic Acids Nucleosome Core Particle Oncogenes Positioning Attribute Procedures Proteins Protocols documentation RNA RNA Conformation Research Resolution Response Elements Roentgen Rays Signal Transduction Structure T-Lymphocyte Techniques Technology Testing Transfer RNA Translation Process Translational Research Untranslated Regions Uridine Visualization X-Ray Crystallography biomacromolecule cancer immunotherapy conformer design high throughput screening improved insight interest manufacture novel novel strategies programmed cell death protein 1 translational potential

项目摘要

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.

我的实验室在几个方面取得了进展。首先，我们开发了一种新的算法和使用 AFM 来研究溶液中 RNA 构象动力学的方法。简而言之，我们现在能够直接可视化溶液中的单个 RNA 构象异构体并确定单个 RNA 分子的结构；计算溶液中 RNA 的总构象空间。 RNA 分子具有高度动态性和构象异质性。这一进展意义重大，因为它使得表征异质构象的单个分子成为可能，例如溶液中的RNA，而不是同质构象的分子集合。我们已经测试、基准测试并应用了我们的新方法和方法来研究溶液中许多重要 RNA 分子的 RNA 结构动力学和构象空间。其中包括 HIV 包装信号 RNA、Rev 反应元件 (RRE) RNA、带/不带 tRNA 配体的 T-box 核糖开关、带/不带配体的钴胺核糖开关 RNA、COVID-19 的 3' 和 5'-UTR RNA 以及RNaseP RNA（全长和核心颗粒）。三份重要的手稿正在审查或待提交。其次，我们证明了使用RNA装置控制和调节小鼠EL4细胞中PD-1基因表达的可行性。 PD-1基因是癌症免疫治疗的关键基因之一。因此，该项目具有转化潜力。基本思想是使用对配体结合做出反应的外部可控 RNA 装置。我们有目的地选择 FDA 批准的配体。这些装置是使用 CRISPR/Cas 9 技术在 T 细胞染色体中设计的。基于去年的进展，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的 PD-1 表达的程序和方案。我们还获得了一些细胞内 RNA 装置的动力学特征信息。我们目前正在使用慢病毒库进行高通量筛选，目的是确定在哺乳动物细胞中既具有高效率又具有理想动力学特性的最佳 RNA 装置。此外，我们在存在和不存在配体的情况下结晶了一种RNA装置，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了解原子水平上配体触发的构象变化，并激发更高效 RNA 装置的新设计。最后，我们在使用高容量 DNA 模板附着珠改进 PLOR 技术（Liu et al., Nat. 2015）方面取得了重大进展。我们的目标是能够合成千碱基长的 mRNA，并在所需位置放置选择性标记或修饰的残基。改进后的 PLOR 的应用之一可以是制造选择性标记有修饰的假尿苷的 mRNA，而不是目前的统一标记，例如辉瑞或 Moderna 的 COVID-19 疫苗中的 mRNA。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Specific labeling: An effective tool to explore the RNA world.

特异性标记：探索 RNA 世界的有效工具。

DOI：
发表时间：
2016-02
期刊：
影响因子：
0
作者：
Liu, Yu;Sousa, Rui;Wang, Yun
通讯作者：
Wang, Yun

Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein.

UHRF1 蛋白识别与异染色质相关的多价组蛋白状态。

DOI：
发表时间：
2011-07-08
期刊：
影响因子：
0
作者：
Nady, Nataliya;Lemak, Alexander;Walker, John R;Avvakumov, George V;Kareta, Michael S;Achour, Mayada;Xue, Sheng;Duan, Shili;Allali;Zuo, Xiaobing;Wang, Yun;Bronner, Christian;Chedin, Frederic;Arrowsmith, Cheryl H;Dhe
通讯作者：
Dhe

RNA conformation: Lightening up invisible states.

RNA 构象：照亮不可见的状态。