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:10702344
- 负责人:
- 金额:$ 256.09万
- 依托单位:
- 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:
- 关键词:AlgorithmsAtomic Force MicroscopyBiochemicalBiologicalBiophysicsCOVID-19COVID-19 vaccineCRISPR/Cas technologyCellsCharacteristicsChromosomesCollaborationsCrystallizationDNADevelopmentDevicesEngineeringEventFDA approvedGene ExpressionGenesGoalsHIVIndividualKineticsKnowledgeLabelLeadLengthLibrariesLigand BindingLigandsMammalian CellManuscriptsMessenger RNAMethodsMolecular ConformationMusNMR SpectroscopyNucleic AcidsNucleosome Core ParticleOncogenesPositioning AttributeProceduresProcessProteinsProtocols documentationRNARNA ConformationResearchResolutionResponse ElementsRoentgen RaysSignal TransductionStructureT-LymphocyteTechniquesTechnologyTestingTransfer RNATranslational ResearchUntranslated RegionsUridineX-Ray Crystallographybasebiomacromoleculecancer immunotherapyconformerdesignhigh throughput screeningimprovedinsightinterestnovelnovel strategiesprogrammed cell death protein 1translational 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, 以及利用AFM研究溶液中RNA构象动力学的方法。简单地说,我们现在 能够直接观察溶液中的单个RNA构象并确定结构 计算单个RNA分子的构象空间;计算溶液中RNA的总构象空间。RNA 分子是高度动态的和构象不均匀的。这一发展意义重大 因为它可以表征异质性的单个分子 构象,例如溶液中的RNA,而不是同质分子的集合 构象我们已经测试,基准和应用我们的新方法和研究方法 RNA结构动力学和构象空间在一些重要的RNA分子, 溶液这些包括HIV包装信号RNA、Rev反应元件(RRE)RNA、 具有/不具有tRNA配体的T-box核糖开关,具有/不具有配体的钴胺素核糖开关RNA,3 '和 COVID-19的5'-UTR RNA和RNaseP RNA(全长和核心颗粒)。三 一些重要的手稿正在审查中或即将提交。二是 证明了使用RNA装置控制和调节PD-1基因的可行性 在小鼠EL4细胞中表达。PD-1基因是癌症的关键基因之一 免疫疗法。因此,这个项目是潜在的翻译。其基本思想是使用 外部可控的RNA装置,其响应于配体结合。我们有目的地 选择FDA批准的配体。这些装置是在T细胞染色体中设计的, CRISPR/Cas 9技术。在去年的基础上,我们建立了 在各种配体浓度下使用以下方法定量PD-1表达的程序和方案 Western和qPCR方法。我们还获得了动力学信息 细胞中一些RNA装置的特性。我们目前正在执行 使用lenti-libraries进行高通量筛选,旨在鉴定最佳RNA装置 在哺乳动物细胞中具有高效和理想的动力学特性。 此外,我们已经在存在和不存在的情况下结晶了一种RNA装置。 配体,从而打开了高分辨率结构测定的大门。值得关注的是 提到任何RNA装置的结构以前从未被确定过。的 RNA的高分辨率结构可能会使我们更好地了解 配体触发的构象在原子水平上发生变化,并刺激更多的新设计 高效的RNA装置。最后,我们在改善《土地注册条例》方面,已取得重大进展 技术(Liu等人,Nat.2015)使用高容量DNA模板附着珠粒。我们的目标是 能够合成具有选择性标记或修饰残基的千碱基长的mRNA 放置在所需的位置。改进的PLOR的应用之一可以是 制造用修饰的假尿苷选择性标记的mRNA,而不是用修饰的假尿苷标记的mRNA。 目前统一的标签,如辉瑞公司的COVID-19疫苗中的mRNA,或 Moderna.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Yun Xing m wang其他文献
Yun Xing m wang的其他文献
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{{ truncateString('Yun Xing m wang', 18)}}的其他基金
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
- 批准号:
8552680 - 财政年份:
- 资助金额:
$ 256.09万 - 项目类别:
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
- 批准号:
8763088 - 财政年份:
- 资助金额:
$ 256.09万 - 项目类别:
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
- 批准号:
8348990 - 财政年份:
- 资助金额:
$ 256.09万 - 项目类别:
NCI X-ray free electron laser (XFEL) initiative
NCI X 射线自由电子激光 (XFEL) 倡议
- 批准号:
10014757 - 财政年份:
- 资助金额:
$ 256.09万 - 项目类别:
NCI X-ray free electron laser (XFEL) initiative
NCI X 射线自由电子激光 (XFEL) 倡议
- 批准号:
10702611 - 财政年份:
- 资助金额:
$ 256.09万 - 项目类别:
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
- 批准号:
7592685 - 财政年份:
- 资助金额:
$ 256.09万 - 项目类别:
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