Using organic chemistry as a tool to explore structures and functions of RNA is the central theme of my proposed research. RNA has the extraordinary ability to perform chemical reactions that are the foundations for life itself . Additionally, scholars continue to debate that the current biochemical milieu of proteins and enzymes evolved from an RNA world . RNA is at the very center of so many biological processes, therefore study of its structure and function is of paramount importance. Each of the following projects houses an RNA centric, fundamentally critical biological process at its core. To unravel the intimate workings of the RNA within each proposed system, a convergent synthesis strategy has been designed to rapidly create a library of analogs or RNA aptamers to be tested in biochemistry assays. In addition to addressing problems with applications to numerous disease states, this proposal has the following merits: 1) there are opportunities for researchers with different levels of experience; each objective could be a collaboration of individual short-term efforts or the sole responsibility of a single multi-semester student. 2) the projects employ equipment common to undergraduate research departments and 3) they are of broad ranging scientific interest allowing for funding opportunities from numerous sources .
I. Structure-based drug design. Chiral transition state analogs as potential new antibiotics.
The growing use of antibiotics in agriculture and medicine has the potential to create a global health care crisis by creating an overwhelming number of antibiotic resistant bacterial strains. Gene transfer from organism to organism, altered uptake of drugs, overproduction of target enzymes and mutated sites of drug binding are all mechanisms bacteria can use to acquire antibiotic resistance. Numerous antibiotics in wide use today specifically target bacterial ribosomes. The ribosome is a monolithic protein-RNA complex responsible for the assembly of proteins in all cells. Despite years of biochemical study and the recent publication of high resolution crystal structures of each subunit and the entire complex of the ribosome, the mechanisms of how some antibiotics inhibit the ribosome's ability to catalyze peptide bond formation remain elusive.
In order to design an effective inhibitor of protein synthesis, some understanding of how the ribosome promotes catalysis must be known. The rate enhancement of peptide bond formation within the ribosome could arise from several possible sources, all of which would contribute to catalysis via a charged tetrahedral intermediate . Designing competitive inhibitors which mimic this intermediate will provide significant advances in antibiotic development.
Chiral phosphorus transition state analogs have been widely used to provide details of the mechanisms of esterase enzymes . Since peptidyl transferase is the reverse reaction of an esterase, the design of the chiral phosphorus inhibitors in this proposal is based on examples from those studies. Figure 1 illustrates the overall framework of analogs that will bind simultaneously to both the amino-acyl (A) and peptidyl (P) tRNA sites within the ribosome and mimic the charged tetrahedral intermediate .
The overall stereochemistry at the phosphorous center is one critical component defining the geometry of the active site. Substituting X = S for X = O on the phosphorus center can test for the presence of a thiophilic metal stabilizing the oxyanion and R1 can be a variable amino acid side chain. Each of these analogs, produced by solid phase synthesis, will be rigorously analyzed for inhibition of peptidyl transferase under multiple turnover conditions using an established in vitro kinetic assay . Additional characterizations can be made using metal ion rescue experiments , chemical mapping and mutagenesis of active site RNA. In vivo inhibition of organisms from various phyla will begin to establish the analogs specificity for bacterial ribosomes and their use as anti-fungals as well. This project is an active collaboration with the laboratories of Scott Strobel and Tom Steitz at Yale University.
II. Conformationally constrained nucleosides: Synthesis of RNA motifs.
Advances in crystallography and biophysical techniques have revealed a myriad of RNA structures other than the canonical A-form helix . These unique structures are present in biologically significant systems and play a role in the formation of functional RNA molecules. Thus, it is important to study the properties of conformational flexibility of RNA nucleosides, because such flexibility makes these structures possible.
In an RNA strand, there are five freely rotating bonds that adjoin two adjacent pentose rings . Rotation about these bonds can result in various degrees of bend, twist and kink in single stranded regions of RNA. RNA structures resulting from the conformers that have been characterized thus far include the U-turn, the tetra loop, bulged G-motifs and, most recently, the kink-turn (K-turn) . From a biochemical standpoint, these unique motifs provide specific sites for RNA-RNA tertiary interactions, metal coordination, protein association and small molecule binding.
The goal of this section is to synthesize conformationally constrained nucleosides and then use them to create pre-organized RNA structures with varying degrees of bend. The first series of analogs (Figure 2) investigate the effects of constraining the position of the 5'-phosphate while preserving the 2'-OH and the flexibility of the base . Both hydrogen bonding with the 2'-OH and the flexibility of the base appear to be necessary to form terminal loop motifs and K-turns . Each analog, synthesized as a fully protected phosphoramidite,
will be suitable for use in solid phase oligonucleotide synthesis.
This way, a library of RNA apatamers can be generated with bends of various degree at selected positions in specific or random sequence contexts. These aptamers can be compared to naturally occurring RNA motifs using a variety of biophysical techniques including gel shift, chemical footprinting, Tm determination, circular dichroism and molecular modeling. Structural similarities may be seen between oligomers containing para-analogs (1p) and naturally occurring, A-form RNAs. Likewise, oligos containing the meta-analog (1m) may adopt a 45° bend as seen in mRNA during translation . The ortho analog (1o) could prove to be the hinge in an RNA mimicking the K-turn.
The synthetically generated RNA structures can be further used to investigate a wide variety of RNA interactions. For instance, RNA-protein interactions will be studied by generating a combinatorial library of peptidomimetics with high affinity for the artificially created K-turns. These high affinity small molecules can be used to identify sequences harboring potential K-turns on both the ribosome and RNAs with unknown structures.
Future studies could focus on RNA-RNA interactions and the mechanics of RNA folding; specifically the folding of the ribosomal RNA into a catalytic moiety.
III. Gene regulation by mRNA-small molecule recognition
Gene regulation in bacteria often occurs by protein factors binding to DNA near the site of the start of transcription. It is also clear that regulation occurs by direct interactions with the mRNA after it has been transcribed . Several models of post-transcriptional regulation suggest the binding of the gene products themselves to regulatory regions (often the 5’ untranslated region) upstream of the start site of translation often attenuates the translation of the gene. The binding of both protein and co-factors to mRNA apparently induces the formation of tightly folded stem-loop structures that sequester the Shine-Dalgarno sequence and initiation codon (Figure 3). This folding affects the initiation of translation of the mRNA into protein [14,15] and thus regulates the formation of the gene product.
What has not been well established at this point is a detailed biochemical model of the RNA motifs that form in the presence of co-factors and a mechanism of discrimination used by these RNA structures to differentiate between potentially very similar affectors. It is the goal of this proposal to study the RNA and affector molecules in the biosynthesis of the vitamin co-factor thiamin from bacteria Rhizobium etli.
As a novel approach to develop a model for the formation of unique RNA motifs that facilitate small molecule interactions leading to the disruption of translation of the thiamin biosynthesis genes, we will employ the techniques nucleotide analog interference mapping (NAIM) and nucleotide analog interference supression (NAIS). The biochemical techniques NAIM and NAIS have been widely used to determine the importance of individual functional groups as well as defining tertiary interactions within large RNA molecules [16,17,18,19]. The data generated from this proposal will add to the current mutagenesis database and further refine critical positions in the regulatory by identifying individual functional groups involved in both inter and intra molecular contacts. A detailed map of these contacts can be generated leading to a three dimentional structure of the active molecule. In addition to the potential of discovering unique RNA motifs involved in gene regulation, the determination of a molecular structure model could have a broader impact on the development of inhibitors or enhancers of this and other gene products.