Joey Shi
Research Mentor: Nils Walter
Mentor Department: Chemistry, LSA
Author(s): Not Available
Session: Session 6 (3:00 PM – 3:50 PM)
Presentation Type: Poster 23
Abstract
Riboswitches are RNA sequences found in the 5’ untranslated region (UTR) of bacterial mRNA. When bound to biological ligands, they can inhibit translation initiation by sequestering the ribosome binding site or trigger transcription termination through the formation of a terminator hairpin. The FMN riboswitch regulates the expression of the essential ribU gene in Clostridium Difficile (C. diff)—a bacterium causing severe colon infections most commonly found in healthcare facilities; as FMN binds to the riboswitch aptamer, it causes a conformational change in the expression platform to promote transcription termination. Antibiotics have greatly improved global life expectancy. However, antibiotic resistance has grown rapidly and is outpacing antibiotic development, creating “superbugs†in bacteria such as C. diff. Hence, I explore riboswitch mechanisms for developing antibiotics, which may offer higher specificity to pathogens and lower side effects in human cells or the surrounding microbiome. Here, we in vitro transcribe RNA Antisense Protein Interactive Drugs (RAPIDs) with two domains: an RNA Polymerase (RNAP) aptamer binding to the RNAP surface, and an antisense oligonucleotide (ASO) binding to the targeted riboswitch and modulating its structure. Using an ASO, we show that we can alter gene expression independently of the presence of FMN and force the formation of the terminator hairpin. We perform experiments using single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) and total internal reflection fluorescence (TIRF) microscopy, monitoring structural changes in single RNA molecules by monitoring the binding of a fluorescently labeled DNA probe to the RNA target. We hypothesize that the binding of the RAPID to the riboswitch-RNAP interface will alter the kinetics of RNA folding, shifting equilibrium towards the stabilization of the terminator hairpin independent of the ligand. Finally, we are developing an in vivo reporter gene assay to evaluate the impact of RAPIDs on gene expression in cellulo. In the fight against rapidly growing antibiotic resistance, this work has the potential to uncover new antibiotics specific to a type of bacterium.
