Research Project
9185116
More than 100 structurally distinct modified nucleosides have been identified, and at least 66 of them occur in eukaryotic cells. However, the functional roles of many other covalent RNA modifications remain poorly characterized or unknown, although they are likely to influence RNA properties and functions such as RNA stability, trafficking, localization, activity, and patterns of interactions with other molecules. Among RNA modifications, m6A is the most prevalent modified base in mRNA. It is a reversible and widespread modification that is primarily located in evolutionarily conserved regions, and is particularly enriched near the stop codon. Identifying m6A residues is challenging. The current m6A mapping approach, methyl-RNA immunoprecipitation and sequencing, involves the immunoprecipitation of ~100-nt-long RNA fragments with m6A-specific antibodies. This approach generates m6A peaks, but it does not identify specific m6A residues. To solve this problem, Dr. Samie Jaffrey?s laboratory at The Weill Medical School of Cornell University successfully developed a novel approach, miCLIP, and they mapped m6A and m6Am residues throughout the transcriptome at single-nucleotide resolution. However, during the development of the miCLIP method, they found that different antibodies can produce very different results and the chosen ones are polyclonal antibodies. There is thus a need for well-characterized recombinant antibodies, specifically optimized for the miCLIP method. In this project, we propose to work with Dr. Jaffrey?s laboratory to develop single-chain variable fragments (scFvs) specifically selected and optimized for this important methodology. In aim 1, we will use the phage display system that has been well established at Mediomics to develop and characterize high affinity scFvs. In aim 2, we will screen for the scFvs that are most suitable for the miCLIP method and then the selected scFvs will be evaluated by Dr. Jaffrey?s laboratory using the miCLIP systems established in his laboratory. Success in the Phase I project will provide us with a strong foundation to develop more needed scFv tools for the detection of other RNA modifications at single nucleotide resolution. We believe that the availability of these sustainable tools will significantly advance research in this important growing field.
