Throwing a wrench in the translational machinery: Discovery of RNA ligands by fluorescence techniques

Ribonucleic acids (RNAs) are underexplored as targets for therapeutics. To date, drug research has focused primarily on protein targets. Inhibition of functional RNA is an alternative approach which can avoid some of the problems of resistance. A few RNAs of interest are the bacterial ribosome, HIV RRE and Tar, human thymidylate synthase mRNA, and the Hepatitis C Virus Internal Ribosome Entry Site. Fluorescently-modified oligoribonucleotides are a relatively cheap method of quickly determining changes in the structure of the RNA. During chemical synthesis, fluorescent modifications can be placed in virtually any position and used to report on local structural changes. This technique was applied to the ribosomal A-site, and expanded to encompass alternative fluorophores and RNA sites. To identify compounds bound to HCV IRES domain IIa, fluorescent and FRET-based constructs were designed. After screening a small compound library in these systems, several were identified as binding. A structure activity relationship based on one class of these binding compounds was determined for the IRES IIa. In order to study the chemical components of RNA binding affinity, fluorescent methods for identifying RNA ligands to two RNA targets were devised. Extension of an oligonucleotide construct based on the ribosomal A site allows monitoring of an additional ligand binding site. FRET-based distance measurements in a viral RNA allow discrimination between functional and nonfunctional conformations, and therefore can be used to identify compounds that stabilize a nonfunctional conformation