RIBOZYMES-- CATALYSIS AND ANTIVIRAL ACTIVITY

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，旨在解决有关RNA生物催化的基本问题，并开发核酶作为选择性工具，用于靶向切割细胞和病毒RNA。在我们证明了工程发夹核酶可以选择性地抑制HIV-1和乙肝病毒复制的合作之后，我们专注于完全在PI的实验室内进行的研究，在这些研究中，我们证明了在稳定表达发夹核酶的BHK-21细胞中抑制了Sindbis病毒。这项建议的具体目的如下：(1)探索一类新型发夹状核酶的活性，这种核酶不需要G位上的切割位点；(2)确定发夹状核酶介导的抑制Sindbis病毒复制的抑制机制、作用位点和序列选择性；(3)评估和优化工程发夹状核酶在哺乳动物细胞内反式切割反应中的酶活性和序列选择性；(4)以Sindbis病毒为模型系统，评估和优化核酶对基因表达的抑制作用。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，我们相信我们将为通过RNA进行的生物化学和靶向RNA失活做出强大而独特的贡献。

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.