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

• 批准号: 10626917
• 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
• 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/10626917
• 关键词: 2019-nCoV; Acceleration; Acylation; Address; Amides; Amino Acid Sequence; Antibodies; Area; Binding; Binding Proteins; Bioinformatics; Biological; Biology; Biomedical Research; Brain Mapping; COVID-19 detection; COVID-19 pandemic; Catalytic Antibodies; Catalytic RNA; Cell surface; Cells; Chemicals; Chemistry; Clinical Laboratory Information Systems; Coronavirus; 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; Licensing; Lysine; Malignant neoplasm of liver; 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; Resected; 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; 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; technology platform; tool; tumor; web-based tool

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

项目成果

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters.

使用人工扩展的碱基对字母表（包括环和错配热力学参数）改进 DNA 结构设计。

• DOI: 10.1101/2023.06.06.543917
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Pham,TuanM;Miffin,Terrel;Sun,Hongying;Sharp,KennethK;Wang,Xiaoyu;Zhu,Mingyi;Hoshika,Shuichi;Peterson,RaymondJ;Benner,StevenA;Kahn,JasonD;Mathews,DavidH
• 通讯作者: Mathews,DavidH

Enzyme-Assisted High Throughput Sequencing of an Expanded Genetic Alphabet at Single Base Resolution.

单碱基分辨率的扩展遗传字母表的酶辅助高通量测序。

• DOI: 10.21203/rs.3.rs-3678081/v1
• 发表时间: 2023
• 期刊: Research square
• 作者: Wang,Bang;Bradley,KevinM;Kim,Myong-Jung;Laos,Roberto;Chen,Cen;Gerloff,DietlindL;Manfio,Luran;Yang,Zunyi;Benner,StevenA
• 通讯作者: Benner,StevenA

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Tau 寡聚化抑制适体的功能选择。

• DOI: 10.1002/anie.202402007
• 发表时间: 2024
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Wang,Bang;Pan,Xiaoshu;Teng,I-Ting;Li,Xiaowei;Kobeissy,Firas;Wu,Zo-Yu;Zhu,Jiepei;Cai,Guangzheng;Yan,He;Yan,Xin;Liang,Mingwei;Yu,Fahong;Lu,Jianrong;Yang,Zunyi;Biondi,Elisa;Haskins,William;Cao,YCharles;Benner,StevenA;Tan
• 通讯作者: Tan