Developing Ultra-High Throughput Sequencing Based Assay For Ligase Ribozymes for the Study of Evolutionary Innovations

The study of evolutionary innovations, or novel traits, is integral to understanding evolution yet is poorly understood. By studying the regions between genotype networks that produce the same phenotype, we can better characterize the process by which innovation occurs. The overarching goal of this study is to assign fitness values to the overlapping genotype network of two catalytic RNA molecules, or ribozymes. Properly characterizing this region requires the study of thousands of individual sequences, which is achievable through the use of high-throughput sequencing analysis. This thesis focuses on developing assays for one of the ribozymes, the ligase ribozyme. Due to the low activity of this ribozyme, the best method for detection proved to be through qPCR amplification and fluorescence detection. This method allows for troubleshooting before sequencing, as well as validation afterward. It was adapted to create the sequencing assay for the ligase ribozyme, which turned out to be comprehensive in its coverage of the tested space, and reproducible between runs. Several ligase sequences were discovered with higher activities than the previous best ligase ribozymes. In addition, the utilization of phased primers proved to be very successful in increasing initial-read diversity in the sequencing pool. The work developing assays for the ligase ribozyme successfully contributed to the larger study on innovation