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:10626917
- 负责人:
- 金额:$ 31.39万
- 依托单位:
- 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:
- 关键词:2019-nCoVAccelerationAcylationAddressAmidesAmino Acid SequenceAntibodiesAreaBindingBinding ProteinsBioinformaticsBiologicalBiologyBiomedical ResearchBrain MappingCOVID-19 detectionCOVID-19 pandemicCatalytic AntibodiesCatalytic RNACell surfaceCellsChemicalsChemistryClinical Laboratory Information SystemsCoronavirusDNADNA biosynthesisDNA sequencingDataDevelopmentDiagnosticDiseaseDisease ManagementDoxorubicinEnzymesEstersEvolutionFeedbackFoundationsGPC3 geneImageIn VitroIndiaInformation SystemsKnowledgeLaboratoriesLanguageLicensingLysineMalignant neoplasm of liverMeasuresMedicalMedical ResearchMedicineModificationMolecular EvolutionNational Institute of General Medical SciencesOrganic ChemistryOutcomePeptidesPerformancePharmaceutical PreparationsPhysiologicalProcessProtein EngineeringProtein Sequence AnalysisProteinsReactionReaction TimeReagentReproducibilityResearch PersonnelResectedRestRibosomal RNARoleScienceShipsSiteSodium ChlorideSpecificitySurgeonSynthesis ChemistrySystemTechnologyTemperatureTestingThermodynamicsToxinTranslatingVisionWorkacyl groupamino groupbiological systemscancer cellcell killingcombinatorial chemistrydesigndrug discoverygene synthesisgenetic informationin vivoinnovationinventionmeetingsmolecular recognitionmultiplex diagnosticsnew technologynext generation sequencingprotein functionsuccesssynthetic biologytechnology platformtooltumorweb-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,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,
目标二。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。
项目成果
期刊论文数量(4)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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:
- 影响因子:0
- 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:
- 影响因子:0
- 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:
- 影响因子:0
- 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
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STEVEN A BENNER其他文献
STEVEN A BENNER的其他文献
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{{ truncateString('STEVEN A BENNER', 18)}}的其他基金
Basic Research for Diagnostics and Surveillance in Lower Resource Environments
低资源环境诊断和监测基础研究
- 批准号:
10669039 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
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:
10478279 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
Basic Research for Diagnostics and Surveillance in Lower Resource Environments
低资源环境诊断和监测基础研究
- 批准号:
10468606 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
Equipment Supplement to 1R01GM141391-01A1 (Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins)
1R01GM141391-01A1 的设备补充(易于使用的试剂盒,用于进化共价标记和灭活蛋白质的试剂)
- 批准号:
10580301 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
PHS2019-02 Omnibus Solic of the NIH, CDC, and FDA for SBIR Apps No Clinical Trial (Parent SBIR R43/4
PHS2019-02 NIH、CDC 和 FDA 的 SBIR 应用程序综合 Solic 尚未进行临床试验(母公司 SBIR R43/4
- 批准号:
10476977 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
Reagents to Chemically Tag Specific Coronavirus Spike Proteins
化学标记特定冠状病毒刺突蛋白的试剂
- 批准号:
10259048 - 财政年份:2021
- 资助金额:
$ 31.39万 - 项目类别:
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