The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and catalyst. The long-term goal of this project is to re-create an RNA world in the laboratory. Towards establishing such a self-replicating RNA system, a catalytic RNA (ribozyme) was developed previously that catalyzes RNA polymerization. However, the polymerization efficiency of this ribozyme is about 100-fold lower than what is required for self-replication, limited by a low substrate affinity. Attempts to improve the efficiency of the polymerase ribozyme by in vitro evolution have so far led to only small improvements. In contrast, recent experiments have shown that hydrophobic cofactors associated with the ribozyme and its substrate are able to improve polymerization efficiency by 3- to 20-fold. This result and the analogy to biological RNAs sparked the idea to search for even better non-RNA cofactors in the form of amino acids and peptides. Combinations of design and in vitro evolution will be used to obtain cofactor dependent ribozymes. Analysis of these ribozymes will help to understand how amino acids and peptides can assist RNAs, on a functional, structural and evolutionary level. Additionally, the recapitulation of life's origin in the laboratory will show how life may have originated during the early history of the planet. The PI is teaching in classroom settings to undergraduates and graduates, is providing research training graduate and undergraduate students in the laboratory, is hosting students from Rancho Bernardo High School in his lab during the summer, and in a pilot project, is mentoring UCSD transfer students from the Thurgood-Marshall College.
The experiments funded in this grant explored, how an early stage of life could have looked like, in a phase called the RNA world, which probably existed on Earth 3.4 - 4 billion years ago. In RNA world organisms, RNA molecules fulfilled the functions that are filled in today's organisms by DNA (genome) and by proteins (most catalysts). A central molecule in an RNA organism would have been an RNA that is able to catalyze the polymerization of RNA, an RNA polymerase. To recapitulate this stage of life in the lab, an RNA with this function was developed previously in the lab. However, this molecule is not efficient enough for the self-replication of a small RNA organism. Our research was focused on improving the efficiency of this RNA polymerase, to facilitate self-replication. Our experiments focused on improving the contacts between the polymerase RNA and its substrate, a partial duplex between two RNAs (the primer and the template). Three different avenues were pursued: First, our most exciting results came from an unexpected discovery: Cofactors on the RNA polymerase that form non-natural base pairs to the substrate, improved substrate binding and polymerization. We will pursue this avenue further by testing other bases, which might have had important functions in an RNA world but which died out when the RNA world was taken over by today's DNA-RNA-protein organisms. Our second line of research focused on effects of the amino acid arginine on polymerization, to facilitate binding of the negatively charged substrate by the positively charged arginine. However, although the arginine was positioned in 10 different positions on the polymerase ribozyme, in none of the 10 positions did the arginine improve polymerization. This suggests that single amino acids may have had only little benefits in an RNA world (in contrast to peptides). Our third line of research, the in vitro selection of improved RNA polymerases, is still in progress. The first two aims led to two publications in peer-reviewed journals. Similarly, all further scientific results will be published in peer-reviewed journals accessible to the scientific community. The experiments were carried out by one postdoctoral researcher, three graduate students, and four undergraduate researchers. Each of them met the PI on a daily basis to design experiments, discuss results, solve technical difficulties, and work on presentations and publications. Our participation in the USTARS program supported the participation of underrepresented minorities by undergraduate research in our lab. The PI participated in mentoring a disadvantaged group of students from the Thurgood-Marshall College, and held two classes at the Helix Charter High School, which has a high ethnic background.
