ABI Innovation: Engineering molecular scissors by computational design with experimental validation

核糖开关是细菌信使RNA分子的一部分，其通过变构来控制基因调控，即，基因是关闭还是开启取决于核糖开关的分子结构，这是由结合事件或“标记物”决定的。核糖开关还已知在一些高等生物（真核生物）中控制选择性剪接，通过确定蛋白质的哪种变体（同种型）由选择性剪接产生，而不是控制蛋白质是否产生。相反，核酶是催化反应的RNA分子，通常催化其自身的切割（顺式）或另一靶分子的切割（反式）。在这个项目中，开发了新的计算方法来设计人工RNA序列，预测形成嵌合的核糖开关/核酶结构，在与另一个小RNA分子杂交时切割自身（顺式），称为“生物”。开发的算法使用约束规划，动态规划和快速傅立叶变换（FFT）。随后通过额外的计算方法选择/优先化待开发的算法返回的序列用于实验验证。最后，最有可能的候选人随后进行实验测试的活性，使用切割测定和在线探测。成功设计的人工核糖开关/核酶RNA分子构成“分子剪刀”，其是静止的（切割关闭），直到被结合事件触发，之后分子是活性的（切割打开）。这项研究的更广泛的影响是科学和教育。由此产生的研究直接有助于合成生物学--纳米技术的“湿”类似物，这两个研究领域都有可能在21世纪改变社会。更广泛地说，这项赠款的研究有助于分子生物学，物理化学和凝聚态物理学领域。这项研究的长期结果可能包括一种对抗艾滋病毒的新方法。人工RNA的计算机设计将有助于我们对分子进化的理解，并将通过提供一种新的方法来寻找某些非编码RNA，如内部核糖体进入位点元件，从而使分子生物学社区受益。这项研究的结果将通过网络服务器和源代码的分发，通过会议和期刊出版物以及会议上的演讲公开。将通过将新的研究成果纳入现有的计算生物学研究生和本科生课程，以及通过在波士顿学院开设新的合成生物学课程，确保在本科生和研究生一级产生教育影响，该学院特别努力从代表性不足的群体中招收学生。为了确保拟议的研究产生广泛的教育影响，每年为期一周的RNA暑期学校将在波士顿学院举行，建立在现有的成功RNA暑期学校的基础上。

Riboswitches are portions of bacterial messenger RNA molecules, which control gene regulation by allostery, i.e., whether a gene is OFF or ON depends on the molecular structure of the riboswitch, which is determined by a binding event or 'trigger'. Riboswitches are also known to control alternative splicing in some higher organisms (eukaryotes), by determining which variant (isoform) of protein is produced by alternative splicing, rather than controlling whether a protein is produced or not. In contrast, ribozymes are RNA molecules that catalyze a reaction, often catalyzing their own cleavage (cis) or the cleavage of another target molecule (trans). In this project, novel computational methods are developed to design artificial RNA sequences, predicted to form a chimeric riboswitch/ribozyme structure, that cleaves itself (cis) upon hybridization to another small RNA molecule, known as a 'trigger'. The algorithms to be developed uses constraint programming, dynamic programming and the fast Fourier transform (FFT). Sequences returned by the algorithms to be developed are subsequently selected/prioritized for experimental validation by additional computational methods. Finally, the most likely candidates are subsequently experimentally tested for activity, using a cleavage assay and inline-probing. Successfully designed artificial riboswitch/ribozyme RNA molecules constitute 'molecular scissors', which are quiescent (cleavage OFF) until triggered by a binding event, after which the molecule is active (cleavage ON). The broader impact of the research in this grant is both scientific and educational. The resulting research contributes directly to synthetic biology -- the 'wet' analogue of nanotechnology, both research areas likely to transform society in the 21st century. More broadly, the research from this grant contributes to the areas of molecular biology, physical chemistry and condensed matter physics. Long-term consequences of this research could include a novel approach to combat HIV. The computational design of artificial RNAs will contribute to our understanding of molecular evolution, and will benefit the molecular biology community by providing a novel approach for finding certain noncoding RNAs such as internal ribosomal entry site elements. Findings from this research will be made publicly accessible through a web server and distribution of source code, by conference and journal publications, and presentations in meetings. Educational impact at the undergraduate and graduate level will be ensured by integrating new research findings into existent graduate and undergraduate courses in computational biology, as well as by developing a new course in synthetic biology at Boston College, which undertakes special efforts to recruit students from underrepresented groups. To ensure a broad educational impact of the research proposed, an annual week-long RNA summer school will be held at Boston College, building on an existing successful RNA summer school.