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，通过变构作用控制基因调控，即基因的关闭或开启取决于核糖开关的分子结构，而核糖开关的分子结构由结合事件或触发事件决定。在一些高等生物(真核生物)中，核糖开关也是已知的控制选择性剪接的方法，它通过确定选择性剪接产生蛋白质的哪种变体(异构体)，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，通常催化自己的裂解(顺式)或另一目标分子的裂解(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，预计将形成嵌合的核糖开关/核酶结构，该结构在与另一小RNA分子杂交时自我切割(Cis)，被称为“触发器”。将要开发的算法使用约束规划、动态规划和快速傅立叶变换(FFT)。由要开发的算法返回的序列随后被选择/优先用于通过附加的计算方法进行实验验证。最后，最有可能的候选者随后被实验测试活性，使用切割试验和内联探测。成功设计的人造核糖开关/核酶RNA分子构成了“分子剪刀”，它是静止的(切割)，直到被结合事件触发，之后分子才活跃(切割开启)。这项拨款研究的更广泛影响既是科学的，也是教育的。由此产生的研究直接促进了合成生物学--纳米技术的“湿”类似物--这两个研究领域都可能在21世纪改变社会。更广泛地说，这笔赠款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。这项研究的长期结果可能包括一种抗击艾滋病毒的新方法。人工RNA的计算设计将有助于我们理解分子进化，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，从而使分子生物学领域受益。这项研究的结果将通过网络服务器和源代码分发、会议和期刊出版物以及在会议上的陈述向公众公布。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课程中，以及在波士顿学院开发一门新的合成生物学课程，将确保在本科生和研究生层面产生教育影响，波士顿学院承担着从代表性不足群体中招收学生的特别努力。为了确保拟议的研究产生广泛的教育影响，波士顿学院将在现有成功的RNA暑期学校的基础上，每年举办为期一周的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.