EVOLUTIONARY ENGINEERING OF ANTIHIV-1 RIBOZYMES

描述：（改编自申请人摘要）。调查人员已 开发了一个实验室系统，用于RNA酶的定向进化。 他们建议使用这个系统来开发最能 切割HIV-1 RNA或DNA内的靶位点，优化活性 在类似于细胞环境的条件下。 的 优化程序进行体外，采用人口 超过1000亿的突变核酶， 重复几轮选择性扩增。每一轮选择性扩增 放大需要1-2天的时间来执行，这样他们就可以调查 在短时间内产生大量的核酶变体。 的 这项研究的目的是开发一个抗HIV-1的家族， 核酶通过使用体外进化方法。 的 申请人对开发核酶特别感兴趣 的 以高特异性和选择性切割双链DNA。 这 将通过修饰I组核酶，使其结合到双- 通过三螺旋相互作用，然后进行 使用一系列DNA底物进行体外进化， 更稳定的双链结构。 DNA裂解组Ⅰ 核酶将被靶向 针对整合酶识别序列 在HIV-1 DNA的U 5末端，针对编码达特的基因 调节蛋白 他们也会产生第二组核酶， 专注于RNA切割反应， 比第一组核酶更具鉴别力。 RNA切割组II 核酶将靶向达特内的高度保守区域 mRNA。 最后，他们将尝试利用E.杆菌 单链结合蛋白反式激活DNA切割组I 核酶，导致抗HIV-1核蛋白的发展。

DESCRIPTION: (Adapted from Applicant's Abstract). The investigators have developed a laboratory system for the directed evolution of RNA enzymes. They propose to use this system to develop ribozymes that are best able to cleave target sites within HIV-1 RNA or DNA, optimizing for activity under conditions that resemble those of the cellular environment. The optimization procedure is carried in vitro, employing a population of well over a thousand billion mutant ribozymes that are subjected to repeated rounds of selective amplification.Each round of selective amplification requires 1-2 days to perform, so that they can survey a very large number of ribozyme variants in a short period of time. The goal of the proposed research is to develop a family of anti-HIV-1 ribozymes through the use of in vitro evolution methodology. The applicants are especially interested in developing ribozymes that cleave double-stranded DNA with high specificity and selectivity. This will be done by modifying a group I ribozyme so that it binds to doubled- stranded DNA through triple-helical interactions, and then carrying out in vitro evolution using a series of DNA substrates that have progressively more stable duplex structure. DNA-cleaving group I ribozymes will be targeted against the integrase recognition sequence at the U5 end of HIV-1 DNA and against the gene encoding the Tat regulatory protein. They will develop group II ribozymes as well, focusing on the RNA cleavage reaction for which they are likely to be more discriminating than group I ribozymes. RNA-cleaving group II ribozymes will be targeted against a highly-conserved region within tat mRNA. Finally they will attempt to exploit the ability of E. coli single-strand binding protein to trans-activate a DNA-cleaving group I ribozyme, leading to the development of anti-HIV-1 nucleoproteins.