Unambiguous transcriptome-wide identification of microRNA:mRNA interactions through enhanced chimeric read amplification

PROJECT SUMMARYProtein synthesis in mammalian cells depends not only on the transcription of RNA from the DNA genomebut also on the stability of that RNA and its rate of translationA class ofnucleotide RNAs known asmicroRNAsplay critical roles in regulating protein synthesis by recognizing complementary sequences in protein coding RNA molecules and causing either RNA cleavage or inhibition of translationMicroRNA regulation plays key roles in nearly every studied human physiological systemand misregulation of individual microRNAs or global microRNA processing has been linked to cancercardiac and kidney diseaseviral infection responseand many other diseasesIdentifying the targets of individual microRNAs provides an essential insight into the functional biological role of each microRNAFurthermoreas functional regulatory molecules microRNAs are now being highly pursued for their use as therapeutic agentswhere the ability to directly map microRNA targets is required to assay both proper on target binding as well as low off target interactionsHowevercurrent methods are lackingas they have either high false positive rateslack the ability to assign targets to individual microRNAsor do not scale to profiling allparticularly low abundancemicroRNAs with quantitative accuracyRecently we developed the enhanced CLIP seqeCLIPmethodologywithfold improved efficiency of generating high throughput sequencing libraries from RBP profiling experimentsenabling highly robust and reproducible RBP target profiling through the incorporation of paired size matched inputsEclipse has successfully developed eCLIP as a highly profitable contract service productand now has an eCLIP kit in beta testingHere we will develop a specialized variant of the eCLIP method for unambiguous mapping of microRNA targets transcriptome wide in the following three aimsValidate unambiguous direct profiling of miRNA targets with chimeric eCLIPchim eCLIPValidate simplified chim eCLIP method for commercializationConversion of chimeric eCLIP into a structured and well documented kit formatEclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and computational biologyparticularly in RNA processing and profiling RNA binding protein targetsThe three aims above will enable microRNA target mapping to be performed in a standard method by all biomedical researchers in academia and industryand create a rigorous standard for validating specificity of therapeutic microRNAsThe ability to properly assess therapeutic miRNA like molecules will provide significant benefits to researchers studying microRNA regulation in various biological contexts and drug companies developing RNA therapies in the clinicAdditionallyin San Diego we are close to many research institutes and biotechs doing RNA research that can provide scientific and commercialization expertise and assistance PROJECT NARRATIVEA class of shortnt RNA molecules known as microRNAs are critical regulators of gene expression from humans to nematodes that act by recognizing complementary sequences in protein coding messenger RNAs and causing either RNA decay or inhibition of translation of those RNAs into proteinIdentification of these microRNA messenger RNA interactions is an essential step towards both a basic research understanding of microRNA regulatory networks as well as towards rigorous validation for the use of microRNAs as therapeuticsThis SBIR proposal seeks to develop improved methods and a kit to directly and unambiguously identify the messenger RNA targets of individual microRNAssolving the current experimental challenges to ensure that accurate identification of these interactions can be performed