RIBOZYMES-- CATALYSIS AND ANTIVIRAL ACTIVITY

核酶——催化和抗病毒活性

• 批准号: 6497254
• 负责人: JOHN MacKenzie BURKE
• 金额: $ 32.85万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497254
• 关键词: RNA virus; Sindbis virus; active sites; antiviral agents; chemical binding; chemical cleavage; chemical stability; complementary DNA; cytotoxicity; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; fluorescent in situ hybridization; gene expression; gene mutation; genetic promoter element; intracellular; mutant; nucleic acid structure; ribozymes; tissue /cell culture; virus replication

The goal of this project is to move quantitative, carefully controlled analysis of RNA enzymes from the test tube into the cell, through a systematic series of experiments designed to address both fundamental issues regarding biological catalysis by RNA, and the development of ribozymes as selective tools for targeted cleavage of cellular and viral RNAs. Following on collaborations in which we demonstrated that engineered hairpin ribozymes can selectively inhibit replication of HIV-1 and hepatitis B virus, we have focused on studies conducted entirely within the PI's lab in which we have demonstrated inhibition of Sindbis virus in BHK-21 cells stably expressing hairpin ribozymes. Specific Aims of this proposal are-(1) Explore the activity of a new class of hairpin ribozymes that do not require G at the cleavage site; (2) Determine the inhibitory mechanism, site of action, and sequence selectivity of hairpin ribozyme-mediated inhibition of Sindbis virus replication; (3) Evaluate and optimize the enzymatic activity and sequence selectivity of engineered hairpin ribozymes in trans-cleavage reactions within mammalian cells; (4) Evaluate and optimize ribozyme inhibition of gene expression using Sindbis virus as a model system. By combining strength and experience in in vitro biochemistry with skills in virology and cell biology, we believe that we are poised to make strong and unique contributions to the fields of biological by RNA and to targeted RNA inactivation.

该项目的目标是通过一系列系统的实验，将定量的、仔细控制的RNA酶分析从试管转移到细胞中，这些实验旨在解决RNA生物催化的基本问题，并开发核酶作为靶向切割细胞和病毒RNA的选择性工具。在合作中，我们证明了工程发夹核酶可以选择性地抑制HIV-1和乙型肝炎B病毒的复制，我们专注于完全在PI实验室内进行的研究，其中我们已经证明了在稳定表达发夹核酶的BHK-21细胞中对辛德毕斯病毒的抑制。本研究的具体目的是：（1）探索一类新的不需要G切割位点的发夹型核酶的活性;（2）确定发夹型核酶介导的辛德毕斯病毒复制抑制的抑制机制、作用位点和序列选择性;（3）评价和优化工程化发夹核酶的酶活性和序列选择性，哺乳动物细胞内的切割反应;（4）使用辛德毕斯病毒作为模型系统评估和优化核酶对基因表达的抑制。通过将体外生物化学方面的实力和经验与病毒学和细胞生物学方面的技能相结合，我们相信我们将为RNA生物学和靶向RNA灭活领域做出强大而独特的贡献。