Computational Design and Experimental Validation of Switchable Ribozymes

目标。今后5年，我提出以下具体、可衡量、可实现的目标：A.核苷酸重复数调节核酶（sRz）。反式作用锤头核酶（HHRz）的计算设计和重复实验测试，该酶选择性地切割具有更多重复数的突变基因的转录本，而不是野生型基因-如引起重复扩展障碍（REDs）的基因。B. Oligo Regulated Ribozymes（核糖传感器）。反式作用锤头核酶的增量设计和测试，有效和快速地切割报告基因转录物，并通过杂交DNA寡核苷酸关闭，可能从不同的致病rna /DNA片段扩增。方法。目标是设计核酶，切割突变转录物，但保持WT完整。然而，这两个转录本通常是相同的，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突变体的“标准”核酶也会与WT结合并切割突变体。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，有可能设计出选择性核酶（sRz）来区分两个不同长度的转录本。这是通过增加核酶的延伸区域来实现的，延伸区域由两个连接器片段组成。我们制作了TriCleaver，一个定制的进化算法，可以自动高效地发现高性能的sRz。同样的进化算法也可以用于设计反式作用的核酶，这种核酶可以靶向任何具有锤头核酶（最好是gus）切割位点的转录物，但这些转录物会被DNA（或实际上）RNA寡核苷酸关闭，并与专门设计的茎II片段结合。结果。a . TriCleaver算法一旦优化，将被整合到我们最新版本的Ribosoft服务器中，并可用于所有人生成各种重复扩展激活的锤头核酶，选择性地靶向导致重复扩展障碍（red）的突变转录本。在未来，这将使我们能够进一步加强与MNIH的合作，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，这可能会导致新的red基因疗法。B. Oligo生物传感器。一旦寡聚调节锤头核酶的原理得到证实，我应该能够与一家感兴趣的生物技术公司（温哥华Bioboost Synbio Consulting inc.）启动合作（并提交Mitacs的资助），以开发taHHRz，在将rtpcr扩增的致病性病毒RNA片段（例如丙型肝炎）结合到taHHRz上时调节报告基因的活性。然后我们将开发致病RNA生物传感器。C.一类锤头核酶裂解效率的预测模型。

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.