UC Irvine

Biochemical properties of ribonucleic acids enable their instrumentality in physiological functions. RNA sequence serves as the intermediary of the genetic code, while secondary and tertiary structures are responsible for RNA’s involvement in regulation and catalysis. As such, discovery and biochemical characterization of RNAs promise to illuminate the relationship between an RNA’s sequence, structure, and functions, thus provide insights into their biological significance. Efforts in functional RNA discovery often turn to in vitro selection or SELEX (systematic evolution of ligand by exponential amplification) to identify RNAs of a functional trait of interest from a diverse pool of sequences. Selected RNA species are subsequently sequenced and characterized for binding affinity or catalytic activity. Given the size and diversity of the enriched pool at the end of a selection, elucidation of RNA sequence and structure remains a labor-intensive process. The work presented here focuses on massive parallel identification and characterization of functional RNAs emerged from in vitro selections through a bioinformatics pipeline known as Apta-Seq.

Enriched sequences from multiple selections were examined via high-throughput sequencing coupled with selective 2´-hydroxyl acylation analyzed by primer extension (SHAPE). These sequences are the output of in vitro selections of RNA aptamers for cyclic-nucleotide monophosphate from genomic DNA libraries. High-throughput analyses of sequences emerged from genomic in vitro selections leads to the discovery of an abundant aptamer with affinity and selectivity towards 3´, 5´-cyclic guanosine monophosphate. Independent structure probing experiments performed on this aptamer produced a binding constant at 5.9 µM and identified key nucleotide residues involved in binding of the ligand. These results established correlation between high-throughput SHAPE data and in-line probing experiments, thus demonstrating Apta-Seq as a sensitive method to massive parallel investigate RNA sequence identity and secondary structure.

Moreover, selection of multi-turnover ribozymes of strain-promoted azide-alkyne catalysis was explored in this work. However, the unsatisfactory output of the selection encourages revision of the selection strategy. Nonetheless, the progress of the selection has the potential to address the challenge of RNA in vivo imaging.