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: 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
• 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

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，用于开发可标记和灭活蛋白质的试剂 应用分子进化基金会 史蒂文·本纳 摘要 根据PAR-19-253，NIGMS寻求新的技术，创造一个积极的反馈回路， 科学通过允许提出新的问题和做出新的发现而向前发展，这反过来又推动了 新技术的发展。30年来，Benner集团为NIGMS的愿景做出了贡献， 开发新技术用于NextGen DNA测序和NextGen DNA合成，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，以及新的 使多重诊断变得简单的平台，今天用于管理COVID大流行的平台。 在这里，我们为NIGMS提供了另一个变革性工具，一个允许研究人员选择 蛋白质靶向和创建试剂（AEGISZyme），化学转化结合的蛋白质。等 40年来一直在寻找试剂，但只取得了有限的成功。 我们将集中于一种转化：将酰基添加到赖氨酸的氨基上的AEGISZymes。 结合的靶标，其中酰化试剂是酯。这种酰化可以使靶蛋白质变性，允许- 让研究人员测试关于该蛋白质在生物学中作用的假设。它可以携带有效载荷，当 与靶蛋白一起内化，携带药物或稳定的AEGISZymes进入细胞。它可以荧光标记 这种蛋白质可以帮助临床医生在切除肿瘤时选择性地切除荧光癌细胞。 为了实现这一变革性和创新性的成果，我们将应用实验室体外进化（LIVE）， 人工扩增遗传信息系统（AEGIS）。该平台将通过满足3个目标来交付： 目标1.我们将使用AEGIS-LIVE来递送AEGISZymes，其通过/失败来酰化靶蛋白中的赖氨酸 反应时间<10秒-1。为了测试这一点，我们将创建这些AEGISZymes，它们使用携带一个 酯基三个目标。将对选定AEGISZymes的发生率进行定量，并对特异性进行度量 针对具有略微不同的氨基酸序列的类似靶标，并且将发现修饰位点， 目标2.我们将使用AEGIS-LIVE提供AEGISZymes，在研究人员选择的靶标上酰化赖氨酸， 周转率，通过/失败周转率>1000，kcat/KM>105 M-1 sec-1。周转率将被衡量 在生理和实验室条件下，并与来自热力学数据的双链体稳定性相关。 目标3.我们将使用AEGIS-LIVE提供在生物系统中稳定的镜像AEGISZymes，包括 运输到细胞中。这将允许AEGISZymes用于生物介质，以支持纳米链毒素 递送，并为在体内使用这些分子奠定基础。 目标4。我们将测试平台的范围，以解决设计参数，如多长时间随机区域 应该是，循环作为完整蛋白质替代物的效果如何，以及如何搜索序列空间。 最后，为了平息任何认为AEGIS-LIVE太“笨重”的观点，我们将创建可分发的工具包， 让他们的接受者制造他们自己的AEGISZymes。这是最终的认证和再现性。