Tuesday, May 21, 2024
5/21/2024
¿ DESCRIPTION (provided by applicant): Long non-coding RNAs (lncRNAs) have recently emerged as regulators of gene expression, functioning via mechanisms that involve chromatin modification, transcription and post-transcriptional processing. LncRNAs exceed the number protein coding genes, are transcribed by the same cellular machinery, and have similar structural features as messenger RNAs. Recent studies have shown that correct orchestration of lncRNA expression is necessary for normal central nervous system development and their dysregulation has been implicated in the etiology of several human neurological diseases. In contrast to advances in lncRNA expression profiling, much less is known about their function. Gaining insight into the mechanisms by which lncRNAs function requires the development of techniques that enable the identification of their genomic targets. One such technique is RNA antisense purification (RAP) which uses a pool of overlapping antisense biotinylated probes to capture lncRNAs and the associated DNA is used to identify the genomic binding patterns. Developed only recently, RAP has been use to profile the genomic targets of a handful of lncRNAs in cultured cell lines. This Phase I SBIR proposal intends to establish the robustness of RAP in neurobiological model systems and to assess its potential for the co-incident identification of lncRNA-associated proteins. This would enable scientists to directly identify which lncRNAs and proteins are both present at a given genomic locus using the same technique. Aim 1 efforts will establish transfer of technical know-how of using RAP to target three lncRNAs whose genomic distribution has been determined in specific cell lines. Aim 2 efforts will establish the feasibility of applying RAP to human and mouse neural cell lines and to perfused mouse brain. The DNA targets of three lncRNAs known to be expressed in brain will be determined by next generation sequencing. To establish whether RAP can be used to isolate and identify lncRNA-associated proteins by mass spectrometry, the lncRNA HOTAIR, which is known to interact with the Polycomb Repressive Complex 2 (PRC2), will be used for proof of concept. PRC2 is a chromatin modifying complex containing the EZH2 and SUZ12 proteins. The goal of Aim 3 is to adapt RAP to enable the successful identification of PRC2 constituent proteins EZH2 or SUZ12 peptides in HOTAIR containing complexes isolated by RAP. Successful completion of these objectives will form the basis of future Phase II efforts where the potential of utilizing RAP in neurological systems will be further developed into a suite of enabling tools (products and services) that will accelerate the functional analysis of lncRNAs in the etiology of neurological behaviors and diseases. The products envisioned include custom synthesis of lncRNA probe sets, RAP assay kits containing a detail protocol and reagents, and providing profiling of lncRNA associated proteins or targeted genomic regions as a service.
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