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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：
10298982
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/10298982
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.

易于使用的试剂盒进化试剂共价标记和灭活蛋白质