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

Boston College, Chestnut Hill MA

Investigators

Abstract

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.