Chaperone-Assisted RNA Crystallography

分子伴侣辅助 RNA 晶体学

基本信息

批准号：
10058842
负责人：
Joseph Anthony Piccirilli
金额：
$ 38.04万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10058842
关键词：
3-Dimensional Acute Affinity Amino Acids Antibodies Antibody Formation Antigens Architecture Bacteriophages Binding Biological Process Cellular biology Chemistry Complex Crystallization Crystallography Development Disease Entropy Evolution Funding Gene Expression Goals Health Human Immunoglobulin Fragments Immunologics Knowledge Laboratories Libraries Macromolecular Complexes Measures Mediating Methods Modeling Molecular Molecular Chaperones Molecular Conformation Molecular Evolution Performance Phage Display Play Population Probability Process Property Proteins RNA RNA Binding RNA Folding RNA Recognition Motif RNA-Binding Proteins Rapid screening Reaction Reagent Research Resolution Ribonucleoproteins Role Scientist Specificity Structural Biologist Structure Surface T7 RNA polymerase Techniques Technology Testing Variant Work X-Ray Crystallography antibody engineering biological systems flexibility improved inorganic phosphate insight macromolecule next generation novel portability protein complex structural biology synthetic antibodies synthetic biology three dimensional structure transcriptome

项目摘要

PROJECT SUMMARY Biological systems possess a highly complex and dynamic cellular RNA population, collectively known as the transcriptome. Many RNAs fold into complex three-dimensional structures, both intrinsically and as ribonucleoprotein (RNP) complexes, and play fundamental roles in nearly every aspect of gene expression. Understanding cell biology, health, and disease requires knowledge of how RNA structure mediates biological function. X-ray crystallography provides a powerful method for structure determination, but RNA crystallization represents a major bottleneck in the process, reflecting in part the limited surface chemistry for mediating lattice interactions and repulsion among the phosphates. Considering the rapid pace of new RNA discovery, there remains an acute need to develop methods to facilitate RNA structure acquisition. For difficult protein targets, antibody fragments (Fab or scFv) have served as effective chaperones for crystallization, and we hypothesized that the large size, conformational properties and surface chemistry of Fabs will facilitate RNA crystallization as well. Using phage-display library selections we demonstrated that Fabs can bind RNA with high affinity and specificity, mediate the majority of lattice interactions in Fab-RNA co-crystals, and provide a molecular replacement model for solving the structures. The long-term goal of this project is to facilitate resolution of the RNA crystallization bottleneck through development of a high-throughput pipeline for antibody production against RNA. The objective of this application is to enable facile access to RNA- binding Fabs and pursue them as reagents for RNA and RNP crystallization and structure determination. To attain this objective we will (a) improve Fab libraries using phage display and molecular evolution approaches to identify amino acid types that tailor complementary determining regions (CDRs) for RNA binding, (b) develop general use crystallization modules with surface and conformational properties adjusted to facilitate crystallization, and (c) use these techniques to create and use Fab complexes of RNA and RNP targets for crystallization and structure determination. Completion of the research will allow facile access to RNA binding Fabs, provide structural biologists with a suite of portable modules for generalized use in RNA/RNP crystallization, and provide important new structural knowledge for understanding biological function.

项目概要生物系统拥有高度复杂和动态的细胞 RNA 群体，统称为转录组。许多 RNA 折叠成复杂的三维结构，无论是本质上还是作为核糖核蛋白 (RNP) 复合物，在基因表达的几乎每个方面都发挥着重要作用。了解细胞生物学、健康和疾病需要了解 RNA 结构如何介导生物功能。 X 射线晶体学提供了一种强大的结构测定方法，但 RNA 结晶是该过程的主要瓶颈，部分反映了有限的表面化学性质介导磷酸盐之间的晶格相互作用和排斥力。考虑到新RNA的快速发展发现后，仍然迫切需要开发促进 RNA 结构获取的方法。对于困难的蛋白质靶标、抗体片段（Fab 或 scFv）已作为结晶的有效伴侣，并且我们假设 Fab 的大尺寸、构象特性和表面化学将促进 RNA结晶也是如此。使用噬菌体展示文库选择，我们证明 Fab 可以结合 RNA 具有高亲和力和特异性，介导 Fab-RNA 共晶中的大部分晶格相互作用，并且提供解决结构的分子替换模型。该项目的长期目标是通过开发高通量管道促进RNA结晶瓶颈的解决用于产生针对 RNA 的抗体。该应用程序的目标是能够轻松访问 RNA- 结合 Fab 并将其作为 RNA 和 RNP 结晶和结构测定的试剂。到为了实现这一目标，我们将 (a) 使用噬菌体展示和分子进化方法改进 Fab 库鉴定适合 RNA 结合的互补决定区 (CDR) 的氨基酸类型，(b) 开发通用结晶模块，其表面和构象特性经过调整以促进结晶，以及 (c) 使用这些技术创建和使用 RNA 和 RNP 靶标的 Fab 复合物结晶和结构测定。研究的完成将有助于更容易地实现 RNA 结合 Fabs，为结构生物学家提供一套可广泛用于 RNA/RNP 的便携式模块结晶，并为理解生物功能提供重要的新结构知识。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Spinach RNA aptamer detects lead(II) with high selectivity.

DOI：
10.1039/c5cc01526j
发表时间：
2015-05-28
期刊：
Chemical communications (Cambridge, England)
影响因子：
0
作者：
DasGupta S;Shelke SA;Li NS;Piccirilli JA
通讯作者：
Piccirilli JA

Structural Basis for Fluorescence Activation by Pepper RNA.

DOI：
10.1021/acschembio.2c00290
发表时间：
2022-07-15
期刊：
ACS CHEMICAL BIOLOGY
影响因子：
4
作者：
Rees, Huw C.;Gogacz, Wojciech;Li, Nan-Sheng;Koirala, Deepak;Piccirilli, Joseph A.
通讯作者：
Piccirilli, Joseph A.

Synthesizing topological structures containing RNA.

DOI：
10.1038/ncomms14936
发表时间：
2017-03-31
期刊：
Nature communications
影响因子：
16.6
作者：
Liu D;Shao Y;Chen G;Tse-Dinh YC;Piccirilli JA;Weizmann Y
通讯作者：
Weizmann Y

Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif.

DOI：
10.1038/s41467-018-06942-3
发表时间：
2018-10-31
期刊：
Nature communications
影响因子：
16.6
作者：
Shelke SA;Shao Y;Laski A;Koirala D;Weissman BP;Fuller JR;Tan X;Constantin TP;Waggoner AS;Bruchez MP;Armitage BA;Piccirilli JA
通讯作者：
Piccirilli JA

Branched kissing loops for the construction of diverse RNA homooligomeric nanostructures