Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins

易于使用的试剂盒可进化出共价标记和灭活蛋白质的试剂

基本信息

批准号：
10478279
负责人：
STEVEN A BENNER
金额：
$ 31.39万
依托单位：
FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
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：
2021-09-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10478279
关键词：
2019-nCoV Acylation Address Amides Amino Acid Sequence Antibodies Area Binding Binding Proteins Bioinformatics Biological Biology Biomedical Research Brain COVID-19 detection COVID-19 pandemic Catalytic Antibodies Catalytic RNA Cell surface Cells Chemicals Chemistry Clinical Laboratory Information Systems DNA DNA biosynthesis DNA sequencing Data Development Diagnostic Disease Disease Management Doxorubicin Enzymes Esters Evolution Feedback Foundations GPC3 gene Image In Vitro India Information Systems Knowledge Laboratories Language Lysine Malignant neoplasm of liver Maps Measures Medical Medical Research Medicine Modification Molecular Evolution National Institute of General Medical Sciences Organic Chemistry Outcome Peptides Performance Pharmaceutical Preparations Physiological Process Protein Engineering Protein Sequence Analysis Proteins Reaction Reaction Time Reagent Reproducibility Research Personnel Rest Ribosomal RNA Role Science Ships Site Sodium Chloride Specificity Surgeon Synthesis Chemistry System Technology Temperature Testing Thermodynamics Toxin Translating Vision Work acyl group amino group base biological systems cancer cell cell killing combinatorial chemistry design drug discovery gene synthesis genetic information in vivo innovation invention meetings molecular recognition multiplex diagnostics new technology next generation sequencing protein function success synthetic biology tool tumor web-based tool

项目摘要

Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins Foundation for Applied Molecular Evolution Steven Benner ABSTRACT Under PAR-19-253, the NIGMS seeks new technologies that create a positive feedback loop that drives science forward by allowing new questions to be asked and new discoveries to be made, which in turn drives the development of new technologies. The Benner group has, for 30 years, contributed to this NIGMS vision, developing new technologies for NextGen DNA sequencing and NextGen DNA synthesis, bioinformatic and evolutionary analyses, dynamic combinatorial chemistry for drug discovery, protein engineering, and new platforms that make multiplexed diagnostics easy, platforms used today to manage the COVID pandemic. Here, we offer the NIGMS another transformative tool, a platform to allow researchers to choose a protein target and create a reagent (an AEGISZyme) that chemically transforms bound proteins. Such reagents have been sought for 40 years with only limited success. We will focus on one transformation: AEGISZymes that add an acyl group to an amino group on a lysine of the bound target, where the acylation reagent is an ester. This acylation may inactivate the targeted protein, allow- ing researchers to test hypotheses about the role of that protein in biology. It may carry a payload which, when internalized with the target protein, carry drugs or stabilized AEGISZymes into a cell. It may fluorescently tag the protein to help clinicians cut away fluorescing cancer cells selectively as they resect a tumor. To achieve this transformative and innovative outcome, we will apply laboratory in vitro evolution (LIVE) to artificially expanded genetic information systems (AEGIS). The platform will be delivered by meeting 3 Aims: Aim 1. We will use AEGIS-LIVE to deliver AEGISZymes that acylate lysines in target proteins with pass/fail reaction times of <10 sec-1. To test this, we will create these AEGISZymes that use a co-substrate carrying an ester group for three targets. Rates of the selected AEGISZymes will be quantitated, specificity will be metricked against similar targets with slightly different amino acid sequences, and modification sites will be found, Aim 2. We will use AEGIS-LIVE to deliver AEGISZymes that acylate lysines on researcher-chosen targets with turnover, with pass/fail turnovers of >1000 and kcat/KM of >105 M-1 sec-1. Turnover rates will be metricked under physiological and laboratory conditions, and correlated to duplex stability from thermodynamic data. Aim 3. We will use AEGIS-LIVE to deliver mirror AEGISZymes that are stable in biological systems, including transport into cells. This will allow AEGISZymes to be used in biological media, to support nanotrain toxin delivery, and to set the stage to use these molecules in vivo. Aim 4. We will test the scope of the platform to address design parameters, such as how long random regions should be, how good loops are as full protein surrogates, and how sequence space is searched. Last, to lay to rest any view that AEGIS-LIVE is too "cumbersome", we will create distributable kits that allow their recipients to make their own AEGISZymes. This is the ultimate in authentication and reproducibility.

轻松使用套件来演化共价标记和灭活蛋白的试剂应用分子进化基础史蒂文·本纳抽象的根据PAR-19-253，Nigms寻求创建积极反馈循环的新技术科学提出允许提出新的问题和新发现，这反过来又驱动新技术的发展。本纳集团（Benner Group 开发用于NextGen DNA测序和NextGen DNA合成的新技术，生物信息学和进化分析，药物发现的动态组合化学，蛋白质工程和新的使多路复用诊断变得容易的平台，如今用于管理Covid大流行的平台。在这里，我们提供了另一个变革性工具，该平台使研究人员可以选择一个蛋白质靶标并创建一种化学转化结合蛋白的试剂（为二酶）。这样的已经寻求40年的试剂，仅取得了有限的成功。我们将重点介绍一种转化：将酰基添加到氨基的赖氨酸赖氨酸的盟约酶结合靶标，其中酰化试剂是酯。这种酰化可能会使目标蛋白质失活，允许 - 研究人员测试有关该蛋白在生物学中作用的假设。它可能会携带有效载荷，当与靶蛋白化合物，将药物或稳定的欺诈酶携带到细胞中。它可能荧光标记该蛋白质可帮助临床医生在肿瘤切除癌细胞时切开荧光细胞。为了实现这种变革性和创新性结果，我们将在体外进化（LIVE）中应用实验室人为扩展的遗传信息系统（AEGIS）。该平台将通过METER 3 AL AIM提供： AIM 1。我们将使用Aegis-live提供均能在具有通过/失败的靶蛋白中酰化赖氨酸反应时间小于10秒。为了测试这一点，我们将创建这些使用携带的共覆盖物酯组的三个目标。选定的柴油的速率将被定量，特异性将被计算针对具有略有不同氨基酸序列的类似靶标，并且会发现修饰位点， AIM 2。我们将使用Aegis-live来提供对研究人员选择的靶标的赖氨酸酶营业额，通过/失败的失误> 1000，kCAT/km> 105 m-1 sec-1。周转率将被大放异彩在生理和实验室条件下，与热力学数据的双链稳定性相关。 AIM 3。我们将使用Aegis-Live来提供在生物系统中稳定的镜像杀菌剂，包括运输到细胞中。这将允许均可在生物培养基中使用，以支持纳米霉素递送，并设定在体内使用这些分子的阶段。 AIM 4。我们将测试平台的范围以解决设计参数，例如随机区域多长时间应该是，循环是完整蛋白质替代物的好环，以及如何搜索序列空间。最后，为了让任何宙斯盾太“笨拙”的观点放弃任何观点，我们将创建可分配的套件来允许他们的接收者制作自己的盟友。这是身份验证和可重复性的最终。