Computational Design and Experimental Validation of Switchable Ribozymes

目标.今后五年，我提出以下具体、可衡量、可实现的目标。A.核苷酸重复序列数目调节的核酶（sRz）。反式作用锤头状核酶（HHRz）的计算设计和重复实验测试，其选择性地切割具有比野生型更大数量的重复的突变基因的转录物-例如引起重复扩增障碍（RED）的那些基因。B。寡核苷酸调节的核酶（核糖传感器）。反式作用锤头状核酶的增量设计和测试，其有效且快速地切割报告基因转录物，并且通过杂交DNA寡核苷酸而被关闭，所述DNA寡核苷酸可能从致病RNA/DNA的不同片段扩增。 方法.目标是设计切割突变转录物但保留WT完整的核酶。然而，这两种转录物通常是相同的，除了（通常）突变体的三核苷酸重复扩增。因此，与突变体结合并切割它的“标准”核酶将与WT结合并切割它。这是不可接受的，因为WT蛋白必须被翻译以避免功能丧失。然而，有可能设计选择性核酶（sRz），可以区分不同长度的两个转录本。这是通过用延伸区扩增核酶来实现的，所述延伸区由两侧为两个接头区段的传感器区段组成。我们精心制作了TriCleaver，这是一种定制的进化算法，可以自动有效地发现高性能的sRz。相同的进化算法可用于设计反式作用核酶，其靶向具有锤头状核酶（优选GUC）的可用切割位点的任何转录物，但使用DNA（或实际上）RNA寡聚物结合到茎II的专门设计的区段而关闭。结果。A. TriCleaver算法，一旦优化，将被纳入我们的Ribosoft服务器的最新版本，并将可用于所有产生各种重复扩增激活锤头核酶，选择性地针对突变体转录，负责重复扩增障碍（RED）。在未来，这将使我们能够进一步加强与MNIH的持续合作，在细胞培养中产生体内结果，显示TriCleaver的效率，特异性和普遍性，这可能导致新的RED基因疗法。B。寡核苷酸生物传感器。一旦寡核苷酸调节锤头核酶的原理得到证明，我应该能够与感兴趣的生物技术公司（Bioboost Synbio Consulting Inc.）开展合作（并提交Mitacs赠款）。in温哥华），用于开发taHHRz，其在结合致病性病毒RNA的rtPCR扩增片段（例如，C型肝炎），至taHHRz。然后我们将开发致病性RNA生物传感器。C.一种锤头状核酶切割效率的预测模型。

Objectives. In the next five years, I propose the following, specific, measurable and realizable objectives. A. Nucleotide-Repeat-Number Regulated Ribozymes (sRz). The computational design and repeated experimental testing of trans-acting hammerhead ribozymes (HHRz) that selectively cleave the transcripts of mutant genes with a greater number of repeats than the wild type- genes such as those causing repeat expansion disorders (REDs). B. Oligo Regulated Ribozymes (Ribosensors). The incremental design and testing of trans-acting hammerhead ribozymes, that efficiently and quickly cleave reporter gene transcripts, and that are switched-off by hybridizing DNA oligos, possibly amplified from distinct segments of pathogenic RNAs/DNAs. Methods. The goal is to design ribozymes that cleave the mutant transcript but leave the WT intact. However, these two transcripts are often identical, except for the (usually) trinucleotide repeat expansion of the mutant. Therefore, a `standard' ribozyme that binds to the mutant and cleaves it will bind to the WT and cleave it as well. This is not acceptable since the WT protein must be translated to avoid loss of function. However, it is possible to design selective ribozymes (sRz) that can differentiate between two transcripts of different lengths. This is achieved by augmenting a ribozyme with an extension region consisting of a sensor segment flanked by two linker segments. We have crafted TriCleaver, a customized evolutionary algorithm that discovers high-performing sRz automatically and efficiently. The same evolutionary algorithm can be used to design trans-acting ribozymes that target any transcript with available cut-sites for hammerhead ribozymes (preferably GUCs), but that are turned-off using a DNA (or indeed) RNA oligo, binding to a specially designed segment of stem II. Outcome. A. TriCleaver Algorithm, once optimized, will be incorporated into the latest version of our Ribosoft server, and will be available for all to generate a variety of repeat-expansion activated hammerhead ribozymes, selectively targeting mutant transcripts, responsible for repeat-expansion disorders (REDs). In the future, this will allow us to further build on our ongoing collaboration with MNIH, to generate in vivo results in cell cultures, showing the efficiency, specificity and generalizability of TriCleaver, which may lead to new genetic therapies for REDs. B. Oligo Biosensors. Once the principle of oligo-regulated hammerhead ribozymes is proven, I should be able to initiate a collaboration (and submit a Mitacs grant) with an interested biotech company (Bioboost Synbio Consulting inc. in Vancouver), for the development of taHHRzs, that regulate the activity of a reporter gene, upon binding of an rtPCR-amplified segment of a pathogenic viral RNA (e.g., hepatetis C), to the taHHRz. We will then be developing pathogenic RNA biosensors. C. Predictive model of cleavage efficiency of one type of hammerhead ribozyme.