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，它通过变异体控制基因调控，也就是说，基因是关闭还是打开取决于核糖开关的分子结构，这是由结合事件或“触发器”决定的。在一些高等生物（真核生物）中，核糖开关也被认为可以控制选择性剪接，通过决定由选择性剪接产生的蛋白质变体（同种异构体），而不是控制是否产生蛋白质。相反，核酶是催化反应的RNA分子，通常催化它们自己的裂解（顺式）或另一个目标分子的裂解（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，预计将形成一个嵌合的核开关/核酶结构，在与另一个小RNA分子（称为“触发器”）杂交时自我切割（顺式）。要开发的算法使用约束规划、动态规划和快速傅里叶变换（FFT）。由待开发算法返回的序列随后被选择/优先排序，以便通过其他计算方法进行实验验证。最后，最可能的候选者随后通过实验测试其活性，使用裂解测定和内联探测。成功设计的人工核糖开关/核糖酶RNA分子构成了“分子剪刀”，在被结合事件触发之前，它们是静止的（切割关闭），之后分子是活跃的（切割开启）。这项资助的研究具有科学和教育两方面的广泛影响。由此产生的研究直接有助于合成生物学——纳米技术的“湿”模拟物，这两个研究领域都有可能在21世纪改变社会。更广泛地说，这项资助的研究对分子生物学、物理化学和凝聚态物理领域做出了贡献。这项研究的长期影响可能包括一种对抗艾滋病毒的新方法。人工rna的计算设计将有助于我们对分子进化的理解，并将为寻找某些非编码rna（如核糖体内进入位点元件）提供一种新的方法，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分发源代码、通过会议和期刊出版物以及在会议上的介绍向公众提供。通过将新的研究成果整合到现有的计算生物学研究生和本科生课程中，以及在波士顿学院开设一门新的合成生物学课程，确保本科和研究生水平的教育影响，该课程将特别努力从代表性不足的群体中招收学生。为了确保拟议研究的广泛教育影响，在现有成功的RNA暑期学校的基础上，每年将在波士顿学院举办为期一周的RNA暑期学校。

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.