Development of Recombinant Antibodies for Detection of RNA Modifications at Single Nucleotide Resolution

More than structurally distinct modified nucleosides have been identified and at least 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 m A 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 m A residues is challenging The current m A mapping approach methyl RNA immunoprecipitation and sequencing involves the immunoprecipitation of nt long RNA fragments with m A specific antibodies This approach generates m A peaks but it does not identify specific m A 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 m A and m Am 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 we will use the phage display system that has been well established at Mediomics to develop and characterize high affinity scFvs In aim 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 RNA modifications have been shown to play a role in intellectual disability microcephaly myoclonus epilepsy depression schizophrenia and addiction relevant behaviors In this project unique recombinant antibodies will be developed characterized and validated for the detection of RNA modifications at single nucleotide resolution The availability of these sustainable tools will significantly advance research in this important growing field