Discovery and analysis of nucleic acid enzymes and aptamers

Functional nucleic acids are DNAs and RNAs that possess unique functions other than cellular information storage and transfer. Among these functional nucleic acids are ribozymes and deoxyribozymes which are DNAs and RNAs that catalyze a variety of reactions such as RNA cleavage through internal transesterification, DNA cleavage, ligation, and even more exotic chemistries such as alkyl or acyl transfer. Another class of functional nucleic acids, aptamers, are ligand-binding DNAs and RNAs that can exhibit exquisite specificity and affinity, and can rival antibodies in biotechnology applications. While many of these DNAs and RNAs were generated using in vitro selection techniques, researchers have found naturally occurring examples that use these functional characteristics to control complex metabolic processes in the cell. These natural metabolite-binding RNAs are termed riboswitches and have proven to be a widespread mechanism of gene control. We used bioinformatics analysis and biochemical characterization to elucidate the structure and function of a new class of riboswitches that binds to purine nucleobases and regulates gene expression in a wide range of bacterial species.; The tight affinity and impressive specificities of both laboratory derived and naturally occurring aptamers make them highly desirable components of biotechnology applications that require molecular receptors, such as affinity purification, compound detection, and clinical diagnostics. Given the considerable potential for aptamer functions, we worked to develop a generalizable in vitro selection technique for producing aptamers. We used in vitro selection to generate RNA-cleaving DNAs that are functional in the presence of divalent metals. These deoxyribozymes were confirmed to be a previously identified catalytic motif called the 8-17 deoxyribozyme. An alternative aptamer generation strategy is proposed that utilizes an aptamer to immobilize a selection population. A series of aptamers were chosen from the literature and analyzed for applicability to our proposed in vitro selection strategy. Ultimately, we generated new classes of cellulose-binding DNAs for use in biotechnology applications such as the allosteric aptamer generation strategy presented here. Additionally, we generated cellulose-binding RNAs using an in vitro selection strategy that incorporated Self Sustained Sequence Replication (3SR) for nucleic acid amplification.