Exploring the Limits of Ribosome Mediated Polymerizations for Expanding the Genetic Code

探索核糖体介导的聚合对扩展遗传密码的限制

基本信息

批准号：
10350599
负责人：
Jaime N Coronado
金额：
$ 2.08万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-25 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10350599
关键词：
Acylation Address Affect Amides Amino Acids Biological Products Biopolymers Biotechnology Catalysis Charge Chemistry Development Diabetes Mellitus Escherichia coli Esters Exhibits Exposure to Fellowship Genetic Code Genetic Transcription Goals Health Human In Vitro Individual Insulin Interdisciplinary Study Libraries Life Mediating Medicine Methods Modeling Molecular Monoclonal Antibodies Nylons Peptides Physical condensation Polymers Procedures Production Proteins Pyrazolones Recombinant Proteins Recombinants Research Ribosomes Savings Scheme Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure System Technology Therapeutic Transfer RNA Translations Treatment Efficacy Universities Vertebral column Work World Health Organization design experimental study innovation medical specialties monomer novel peptide drug polymerization polypeptide scaffold synthetic biology therapeutic protein tool

项目摘要

PROJECT SUMMARY Recombinant protein production (RPP) has become a powerful tool for producing life-saving therapeutics such as insulin, monoclonal antibodies, and other critical biopharmaceuticals. However, this promising technology is severely limited by the ability to efficiently expand the genetic code to incorporate exotic monomers and backbones for enhanced therapeutic function. The vast majority of biopolymers currently produced by the translation machinery display polyamide backbones; therefore, the possible secondary and tertiary confirmations available to proteomimetics synthesized via RPP are limited to these scaffolds. Since monomer sequence defines structure and structure defines function, expanding the available monomer pool for translation will produce biopolymers with greater structural complexity and thus increase the functional capabilities for proteomimetic therapeutics. A major limitation to addressing this issue is the underexplored capability of the ribosome to incorporate unnatural monomers, especially those that do not form peptide (amide) bonds. Toward this goal, this proposal aims to develop genetically encoded chemistries that can be catalyzed by the ribosome to synthesize sequence defined polymers (SDPs) with structurally diverse backbones (non-peptide bonds). Specifically, I will design and synthesize a library of a-hydrazino-keto ester monomers, charge them onto orthogonal tRNA, introduce them to the translation machinery in vitro, and evaluate the ability of the ribosome to catalyze their polymerizations. The hydrazine and keto ester moieties are known to react in solution to form various heterocyclic products. Importantly, the last mechanistic step in heterocyclic formation is amide bond formation, a specialty of the ribosome. Therefore, I hypothesize that bifunctional monomers comprised of both these moieties are capable of forming heterocyclic linkages via ribosome mediated catalysis. Encouraging preliminary results by our collaborative and interdisciplinary research team have suggested this goal is achievable as we have found the ribosome to be more accommodating than previously thought. The experiments in this proposal will (1) broaden our understanding of molecular translation, (2) elucidate the limitations and principles that govern genetic code reprogramming, and (3) expand the synthetic toolbox for the development of biologically derived SDPs via the ribosome. Accomplishing the aims in this proposal will increase the backbone diversity currently attainable by the translation machinery and could produce SDPs that might exhibit greater therapeutic efficacy.

项目概要重组蛋白生产（RPP）已成为生产拯救生命的有力工具胰岛素、单克隆抗体和其他关键生物制药等治疗药物。然而，这有前途的技术受到有效扩展遗传密码以整合的能力的严重限制用于增强治疗功能的外来单体和主链。目前绝大多数生物聚合物由翻译机械生产的显示聚酰胺主链；因此，可能的次要和通过 RPP 合成的蛋白质模拟物可用的三级确认仅限于这些支架。自从单体序列定义结构，结构定义功能，扩展可用单体池翻译将产生结构更复杂的生物聚合物，从而增加功能蛋白质模拟疗法的能力。解决这个问题的一个主要限制是尚未充分探索核糖体掺入非天然单体的能力，尤其是那些不形成肽的单体（酰胺）键。为了实现这一目标，该提案旨在开发可以由核糖体催化合成结构多样的序列定义聚合物（SDP）主链（非肽键）。具体来说，我将设计并合成一个α-肼基酮酯单体库，将它们充电到正交 tRNA 上，将它们引入体外翻译机器，并评估翻译机器的能力核糖体催化它们的聚合反应。已知肼和酮酯部分发生反应溶液生成各种杂环产物。重要的是，杂环形成的最后一步是酰胺键的形成，是核糖体的一个特点。因此，我假设双功能单体由这两个部分组成的能够通过核糖体介导形成杂环键催化。我们的协作和跨学科研究团队取得了令人鼓舞的初步成果表明这个目标是可以实现的，因为我们发现核糖体比以前更适应想法。本提案中的实验将（1）拓宽我们对分子翻译的理解，（2）阐明控制遗传密码重编程的局限性和原则，以及（3）扩展合成用于通过核糖体开发生物衍生 SDP 的工具箱。实现本次目标该提案将增加翻译机构目前可实现的骨干多样性，并且可以产生可能表现出更大治疗效果的 SDP。