Genetic circuits based on allosteric ribozymes

基于变构核酶的遗传电路

• 批准号: 7346991
• 负责人: Andrew D Ellington
• 金额: $ 26.51万
• 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 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7346991
• 关键词: Biological Assay; Biological Models; Biosensor; Catalysis; Catalytic DNA; Catalytic RNA; Cells; Cleaved cell; Cyclic AMP; Cyclic AMP Receptor Protein; Emulsions; Escherichia coli; Gene Expression Regulation; Genetic; Glucose; Growth; In Vitro; Introns; Lac Operon; LacZ Genes; Lactose; Ligands; Ligase; Ligation; Messenger RNA; Methods; Nucleic Acids; Oils; Operon; Production; RNA Splicing; Reaction; Repression; Study models; Technology; Water; base; catalyst; gene therapy; genetic regulatory protein; in vivo; novel; small molecule; sugar; synthetic biology; technology development; tool

DESCRIPTION (provided by applicant): Allosteric nucleic acid catalysts can directly transduce ligand recognition into catalysis, and thus are particularly useful as biosensors. We have previously developed a variety of aptazymes, based on ribozyme and deoxyribozyme ligases and Group I self-splicing introns, and have also developed novel assay formats for these aptazymes. Now, we propose to adapt aptazymes to function in vivo, in order to build genetic circuits that can control gene regulation. In particular, we propose to replace or augment regulatory proteins with regulatory aptazymes in a particularly well-studied model system, catabolite repression in E. coli. In greater detail: Specific Aim 1. In vitro selection of aptazymes dependent on cAMP and CRP. The small molecule cAMP and cAMP receptor protein (CRP) are instrumental in modulating the expression of operons involved in the utilization of sugars, such as the lac operon. Starting with three different ribozyme platforms (hammerhead, L1 ligase, and Group I self-splicing intron) we will select and characterize aptazymes that are activated by cAMP or CRP. It should be noted that a cAMP-dependent hammerhead aptazyme is already available, and thus that Specific Aims 2 and 3 do not completely rely upon the execution of Specific Aim 1. Specific Aim 2. Aptazyme optimization for multiple turnover and in vivo function. The aptazymes selected as a result of Specific Aim 1 will be further optimized for multiple turnover reactions using a novel trans-selection method based on in vitro compartmentalization (IVC) in water-in-oil emulsions. The fastest aptazymes will then be further screened for function in E. coli cells. We believe that both of these technologies will ultimately be necessary to identify aptazymes that are highly efficient in vivo. The IVC method will allow us to screen larger pool sizes prior to assessing smaller, functional pools in vivo. Specific Aim 3. Constructing genetic circuits with aptazymes. The aptazymes from Specific Aims 1 and 2 will be activated by either cAMP or CRP, and will be able to either cleave, ligate, or splice either Lacl or LacZ mRNAs. They thus represent modular tools for the construction of a variety of genetic circuits for catabolite repression. We will generate several such circuits in which the addition of glucose leads, for example, to the cleavage of Lacl mRNA or to the ligation of LacZ mRNA. These circuits will be further evolved for in vivo function by iterative growth in the presence of glucose and lactose. The development of technologies for the production of aptazymes and genetic circuits that can function in vivo should potentiate the construction of organismal biosensors, regulatable gene therapies, and new tools for the burgeoning field of synthetic biology.

描述（由申请人提供）：变构核酸催化剂可以将配体识别直接转导为催化，因此作为生物传感器特别有用。我们以前曾基于核酶和脱氧核酶和I组自剪接的内含子开发了多种幻想，并为这些合适性开发了新颖的测定格式。现在，我们提议适应适体在体内发挥作用，以构建可以控制基因调节的遗传回路。特别是，我们建议用特别良好的模型系统（大肠杆菌中的分解代谢产物抑制作用）中的调节齐扎斯代替或增强调节蛋白。更详细地：具体目的1。依赖于CAMP和CRP的适量齐的体外选择。小分子cAMP和CAMP受体蛋白（CRP）在调节参与使用糖（例如LAC操纵子）的操纵子的表达方面发挥了作用。从三个不同的核酶平台（Hammerhead，L1连接酶和I组自剪接内含子）开始，我们将选择并表征被CAMP或CRP激活的Aptazymes。应当指出的是，依赖于cAMP的锤头aptazyme已经可用了，因此，特定的目标2和3并不完全依赖于特定目标1的执行。特定目标2。aptazyme优化多个失误和体内功能。根据特定目标1所选择的适量酶，将使用基于体外隔室化（IVC）在油中乳液中的新型反选择方法进一步优化多个离职反应。然后，将最快的适物酶进一步筛选出在大肠杆菌细胞中的功能。我们认为，这两种技术最终都是必要的，以识别体内高效的趋势。 IVC方法将允许我们在评估体内较小的功能池之前筛选较大的池尺寸。特定的目标3。用忠实者构建遗传回路。来自特定目标1和2的Aptazymes将被CAMP或CRP激活，并且能够裂解，结扎或拼接LACL或LACZ mRNA。因此，它们代表了用于构建各种遗传回路以进行分解代谢物抑制的模块化工具。我们将产生几个这样的电路，其中添加葡萄糖导致LACL mRNA的裂解或LACZ mRNA的连接。通过葡萄糖和乳糖的存在，这些电路将通过迭代生长而进化为体内功能。可以在体内发挥作用的适词和遗传回路的技术的开发，应增强有机生物传感器，可调节基因疗法的结构，以及用于合成生物学迅速发展领域的新工具。