NIH is seeking innovative methods for highly parallel detection of ligand/target binding, to develop a database of the biological activities of small organic molecules. Ribonucleic acids (RNAs) are involved in many critical aspects of normal cellular function, genetic disorders, and microbial infection. Like proteins, RNAs achieve these biological functions by adopting intricate, three-dimensional folds and architectures that can participate in recognition and binding. Recent studies have shown that small ligands can modulate RNA function in many ways; and interest in RNA as a drug target is rapidly growing. The long-term goal of the proposed SBIR is to develop a flexible 'mix-and-measure' nanowell plate assay for the highly parallel analysis of RNA-ligand binding interactions. On Phase I, HIV-1 TAR RNA and the A site of Escherichia coli 16S ribosomal RNA will be used to show that the proposed assay will determine whether binding took place, and produce information on the impact that the interaction had on the chemistries of the ligand and/or RNA. RNAs commonly undergo posttranscriptional modifications; therefore, Phase I will demonstrate the ability to detect the impact that modifications have on RNA-ligand binding interactions. Phase I will show that the proposed assay can be used with diverse small RNAs, and even with the 16S ribosomal RNA, the 30S ribosomal subunit, and the whole bacterial ribosome, with little or no methods development. Phase I will also show that, due to the highly specific nature of the data that is produced, the results will be very reliable, with many potential sources of false responses eliminated. Finally, studies will show that the proposed approach has the potential for exquisite sensitivity and very high-throughput. The proposed platform can be used for high-throughput screening of synthetic chemistry and natural product libraries, to assess their biological activities. Active compounds identified by the screens can, in turn, be used as research tools elucidate biological processes through chemical genetic approaches, or can form the basis of therapeutics or imaging agent development programs. In addition, the platform can be used to determine the spectrum of activity of a drug lead and/or its potential toxicity, to expedite drug discovery and development, and minimize risk
