Generating monoclonal antibodies that selectively and specifically recognize epitranscriptomic modifications.

ended

Epitranscriptomic modifications constitute specific marks in mRNA that regulate gene expression. Identification of epitranscriptomic modifications requires next generation sequencing analyses and specific validation, processes that are cumbersome and time consuming. Furthermore, while some modifications are straightforward to identify, as they cause specific base changes (like RNA editing), most are only detectable either through chemical means (requiring large quantities of material) or by immunoprecipitation followed by sequencing. Of these techniques, immunoprecipitation requires far less material and can be used in any molecular biology laboratory, but depends exquisitely on the quality of the monoclonal antibodies that recognize the modification - their specificity, selectivity and sensitivity. Most anti-modification antibodies are generated using traditional methods for the production of monoclonals to small molecule haptens (e.g. conjugation to a large protein carrier and injection into animals together with an adjuvant). While these methods can generate anti-hapten responses with some specificity they are generally not very selective. We have developed a novel immunization platform that exquisitely focuses the antibody response to a hapten to generate high affinity monoclonals with a high degree of specificity. We propose to employ this approach to generate anti-inosine antibodies (for the detection of editing in disease contexts); anti-m6A antibodies (which would be benchmarked against the ones currently in use, validating our approach); and antipseudo-uridine antibodies (a tool that is lacking in the field and for which traditional approaches have completely failed). The generation of specific and selective antibodies to these three modifications, will open the way toward a better understanding of their intricate molecular roles in biological proceGenerating monoclonal antibodies that selectively and specifically recognize epitranscriptomic modifications.