Chaperone-Assisted RNA Crystallography

项目总结 生物系统拥有高度复杂和动态的细胞RNA种群，统称为 转录组。许多RNA折叠成复杂的三维结构，既有内在的也有AS的 核糖核蛋白(RNP)复合体，在基因表达的几乎每一个方面都发挥着重要的作用。 要了解细胞生物学、健康和疾病，需要了解RNA结构如何调节生物 功能。X射线结晶学为确定结构提供了强有力的方法，但RNA 结晶是这一过程中的一个主要瓶颈，这在一定程度上反映了 调节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 尽管发现了RNA结构，但仍然迫切需要开发促进RNA结构获取的方法。对于困难的人 蛋白质靶标、抗体片段(Fab或ScFv)被用作结晶的有效伴侣，以及 我们假设，Fabs的大尺寸、构象特性和表面化学将有助于 RNA结晶也是如此。通过噬菌体展示文库的选择，我们证明了Fabs可以与RNA结合 具有高亲和力和特异性，介导了Fab-RNA共晶体中的大部分晶格相互作用，以及 提出了解决结构问题的分子置换模型。这个项目的长期目标是 通过开发高通量管道来促进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方便地访问RNA- 结合Fabs，并将其作为RNA和RNP结晶和结构确定的试剂。至 为了实现这一目标，我们将(A)利用噬菌体展示和分子进化方法来改进Fab文库 确定为RNA结合量身定做互补决定区(CDR)的氨基酸类型，(B) 开发表面和构象属性可调整的通用结晶模块，以便于 结晶，以及(C)使用这些技术来创建和使用RNA和RNP靶标的Fab复合体 结晶和结构测定。这项研究的完成将使人们能够方便地获得RNA结合 FABS为结构生物学家提供了一套可广泛用于RNA/RNP的便携式模块 结晶，为理解生物功能提供了重要的新的结构知识。

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.