A high-throughput kit for detection of RNA modifications

DESCRIPTION provided by applicant The broad objective of this project is to develop an integrated wet lab dry lab kit for high throughput detection of RNA modifications The final product will provide all required molecular biology reagents a set of simple and robust experimental manipulations for facile analysis of modifications and researcher friendly analytic cloud based software requiring no special bioinformatics skills or computing hardware Emerging data suggests that post transcriptional modifications of many types of cellular RNA play an important and largely unexplored role in biology and in development and maintenance of the nervous system In particular a notable variety of neural diseases are due to specific defects in RNA modification including autosomal recessive intellectual disability ARID non syndromic X linked mental retardation myoclonus epilepsy ragged red fibers MERRF mitochondrial encephalopathy lactic acidosis with stroke like episodes MELAS and familial dysautonomia Initial evidence for the links between RNA modification regulation and gene expression mRNA splicing nuclear export and protein translation suggest much broader unknown effects given that modification states for most RNAs in most cell types are not well defined High throughput RNA sequencing has revolutionized the study of gene expression but it has not been widely applied to identifying most types of RNA modifications due to lack of methods for detection of modifications that are suitable for commercial use and lack of methods for complex post sequencing analytics This Phase proposal focuses on developing such methodology for three types of biologically relevant but relatively understudied RNA modifications with the following Specific Aims I Develop methods for identifying RNA sites with m A m C or andapos O methylation II Develop an analysis pipeline to identify sites with andapos O methylation m A or m C To achieve these aims a novel biochemical approach will be employed for detecting and mapping andapos O methylation leveraging a known chemical reaction coupled with ligation methods to enrich for these sites A distinct novel approach will be applied
to detect m A and m C modifications by differential analysis of reverse transcription between enzymatically treated samples An integrated cloud based platform with custom analytic pipelines will be developed for processing and interpreting the RNA sequencing data to simplify complex analyses required to identify modifications with precision Together these all inclusive kits will form both pre and post sequencing means for large scale RNA modification detection with turn key ease of use

PUBLIC HEALTH RELEVANCE This project will advance research on RNA modifications a key type of processing events that plays an important role in the development of the nervous system and many neurological diseases Researchers will be able to use the tools developed by this work to detect multiple types of RNA modifications in the cells of human and other model species using a high throughput robust commercial kit Broad application of this technology should significantly advance the knowledge of RNA modification and its many effects on gene regulation and human health