Ribozymes for Peptide- and Protein- Sensing Chip Arrays

用于肽和蛋白质传感芯片阵列的核酶

• 批准号: 6658132
• 负责人: Andrew D Ellington
• 金额: $ 23万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6658132
• 关键词: allosteric site; biosensor device; biotechnology; ligands; ligase; microarray technology; molecular shape; posttranslational modifications; protein engineering; proteomics; ribozymes; synthetic enzyme; technology /technique development

This grant proposal explores the development of a nascent and novel technology, effector-activated ribozymes, also known as aptazymes. In vitro selection techniques have previously yielded a wide variety of nucleic acid binding species, aptamers (B.1). Aptamers can be appended to ribozymes to yield ligand-dependent, allosteric catalysts (B.2). It may be possible to use the broad-ranging molecular recognition abilities of aptamers to generate a similar range of aptazymes. Since aptazymes in effect transduce molecular recognition into catalysis, they can potentially be used as biosensors. We propose to develop aptazyme arrays that can be used to detect and quantitate proteins in organismal proteomes. In particular, by developing aptazymes that can recognize peptide epitopes and protein targets it may prove possible to readily create both targets and biosensors for array construction (B.3.). As a starting point for these goals we have chosen to use a small ribozyme ligase (L1) that was selected in our laboratory (C.1.). The L1 ligase has already been engineered to be responsive to oligonucleotide and small molecule effectors, and the allosteric activation parameters of the engineered variants are far superior to their protein counterparts (C.2-C.4). However, the ability to engineer the L1 ligase to be responsive to peptide or protein effectors should greatly potentiate the development of novel proteome chips. To this end the development efforts for the L1 ligase fall into two major areas: (1)Adapting the L1 ligase to be activated by peptide ad protein effectors (D.1.-D.3), and (2)Adapting peptide and protein-activated aptazymes to function in chip arrays (D.4).

该赠款提案探讨了一种新生和新型技术的发展，效应子激活的核酶，也称为Aptazymes。 体外选择技术先前已经产生了多种核酸结合物种适体（B.1）。 可以将适体附加到核酶中，以产生配体依赖性的变构催化剂（B.2）。 可以使用适体的广泛分子识别能力来产生类似的敏锐范围。 由于实际上是将分子识别转化为催化的，因此它们可能被用作生物传感器。 我们建议开发可用于检测和定量有机体蛋白质组蛋白质的合适阵列。 特别是，通过开发可以识别肽表位和蛋白质靶标的丙唑，可以很容易地创建靶标构建靶标和生物传感器（b.3。）。作为这些目标的起点，我们选择使用在我们实验室中选择的小核酶连接酶（L1）（C.1。）。 L1连接酶已经设计为对寡核苷酸和小分子效应子的反应，并且工程变体的变构激活参数远远优于其蛋白质对应物（C.2-C.4）。 但是，设计L1连接酶对肽或蛋白质效应子有反应的能力应大大增强新型蛋白质组芯片的发展。 为此，L1连接酶的开发工作属于两个主要领域：（1）调整L1连接酶被肽AD蛋白效应子（D.1.-D.3）激活，以及（2）适应肽和蛋白质激活的丙烷的功能，以在芯片阵列中（D.4）中的功能。