Influence of Biological Small Molecules on the Twister Ribozyme and Development of Methodologies to Study and Discover Small Riboyzmes.

다국어 초록 (Multilingual Abstract)

The central dogma of molecular biology describes the distinct roles for the three major biological macromolecules: DNA, RNA and protein. In recent years, it has become increasingly apparent that the roles for DNA, RNA and proteins are more complex and less distinct than previously thought. This is especially true for RNA which has been found to be involved in a plethora of different biological activities. Of particular interest are functional RNAs such as riboswitches, RNA which undergo structural changes upon ligand binding, and ribozymes, RNAs which exhibit catalytic activity.Ribozymes are a relatively recent discovery with the earliest examples identified in the 1980’s with the discovery of the self-splicing RNA intron in Tetrahymena thermophila. Since then, a number of ribozymes have been identified among diverse organisms. Ribozymes are divided into two main classes, the large and small ribozymes, with a majority of ribozymes belonging to the small ribozyme class. The small ribozymes are typically 100 nucleotides or fewer in length and catalyze a site-specific phosphodiester cleavage reaction. A total of nine small self-cleaving ribozymes have been identified thus far with nearly half being identified in the last several years. Among the newly identified self-cleaving ribozymes is the tc, which has been intensely studied through several structural and mechanistic studies to elucidate its catalytic mechanism.The twister ribozyme is one of the most catalytically active self-cleaving ribozymes as it is purported to use multiple catalytic strategies. The twister ribozyme is hypothesized to utilize two ionizable residues, with pKas of 6.9 and 9.5, thought to be a general acid and general base. The higher pKa of 9.5 has been attributed to a guanine, which is hypothesized to serve as both a general base and electrostatically stabilize the phosphorane intermediate. The lower pKa of 6.9 has been attributed to the conserved A1 which is purported to act as a general acid. While crystallographic evidence supports the role of A1 as a general acid, the available experimental evidence is mixed with no clear answer. As such, one goal of this thesis is to further define the catalytic mechanism of the twister ribozyme. The other goal of this thesis is to develop new methodologies to investigate the structure and biological activity of small self-cleaving ribozymes.Chapter 2 is focused on characterizing and establishing the mechanism by which small biological molecules stimulate the self-cleavage activity of the twister ribozyme. We find that moderate buffer concentrations can stimulate the catalytic activity of the twister ribozyme up to 5-fold. The buffers are a range of small molecules including common laboratory buffers, and biological metabolites such as imidazole, amino acids, and amino sugars. Additionally, Bronsted plot analysis indicates that the small molecules assist in proton transfer, most likely general acid catalysis. Further, we observe that at biological Mg2+ concentrations and low pH, the self-cleavage activity of the twister ribozyme appears largely buffer independent while at biological Mg2+ concentrations and pH or at high Mg2+ concentrations, the self-cleavage activity of the twister ribozyme is buffer dependent. As such, we propose a multi-channel mechanism for the twister ribozyme consisting of a buffer independent and buffer dependent channels. This work establishes a simple way to overcome the limited chemical diversity of RNA and could apply to the catalytic mechanisms of many ribozymes in vivo.Chapter 3 is aimed at characterizing the role that the A1 residue plays in the catalytic mechanism of the twister ribozyme through a combination of chemical rescue and glycosidic conformational analysis experiments. We observe that inhibited twister ribozyme constructs containing an A1 N3 deaza or abasic A1 modification can be rescued over 100-fold using small protonatable molecules such as imidazole and histidine, similar to the chemical rescue effects observed in the antigenomic HDV ribozyme with a C76U mutation. Additionally, Bronsted plot analysis indicates that the small molecules rescue catalytic activity through proton transfer, suggesting that the wild type A1 residue is also involved in proton transfer, likely general acid catalysis. We also determine through glycosidic conformational analysis that an 8BrA1 modified twister ribozyme is up to 10-fold faster than a non-modified A1 residue in an appropriate background suggesting that the catalytic conformation is syn as suggested by multiple crystallographic studies. This study provides functional evidence that A1 is syn while conducting proton transfer.The goal of Chapter 4 is to develop a novel computational and experimental pipeline to identify and assay the in vitro catalytic activity of putative ribozymes en masse. To do this, we developed a computational pipeline, based on RNABOB, to identify ribozyme candidates of known ribozyme motifs with variant secondary structures. Four RNABOB descriptors were written based on the type III hammerhead ribozyme, the human HDV-like CPEB3 ribozyme and the type P1 twister ribozyme, both with and without the P3 stem, with loosened constraints identified nearly 23,500 ribozyme candidates among 18 different organisms. Additionally, we optimized an experimental pipeline to assay thousands of ribozymes at a time for in vitro catalytic activity by taking advantage of massively parallel oligo synthesis (MPOS) to produce the DNA templates. Currently, we have optimized a majority of the experimental pipeline and successfully identified all active ribozymes in a sample set of oligos.The goal of Chapter 5 is to develop a scaffold to artificially increase the size of small nucleic acid structures to a size that is amenable for cryo-EM visualization and single particle reconstruction. Thus far, we have developed a nucleic acid-based scaffold, dubbed a “Nanosprout Scaffold,” that can multimerize small nucleic acids into a larger structure of an appropriate size for cryo-EM visualization. The Nanosprout Scaffold consists of a DNA oligonucleotide, denoted DNA guide, that multiple nucleic acids of interest with 5’-extensions can multimerize to via complementary base pairing interactions. So far, we have designed multiple Nanosprout Constructs based on the env22 twister ribozyme, and the 10MD5/10MD5- AC DNAzymes with moderate success. We observe, in aqueous conditions, that Nanosprout Constructs assemble with moderate to high affinity and with catalytic activity approaching, or on par, with the native constructs. Additionally, we are able to visualize individual env22 twister ribozymes via TEM and cryo-EM, albeit several issues persist as the fully multimerized env22 Nanosprout Constructs either partially disassemble or do not form under these conditions. The Nanosprout Scaffold is a promising start to a scaffold system that can be adapted to small nucleic acids for cryo-EM structure determination.