Single-molecule microscopy (SiM-KARTS) and chemical probing (SHAPE) of lncRNA

Long noncoding RNAs (lncRNAs) provide a wealth of potential as therapeutic targets and diagnostic biomarkers. Several heart-specific lncRNAs have been identified, including ones that play important roles in heart health and disease. Despite their importance, the mechanisms by which these lncRNAs affect the heart have not been dissected at the molecular level. We propose to utilize single-molecule fluorescence techniques to determine the structure and molecular mechanism of a heart-specific lncRNA, Myheart (Mhrt), which protects the heart under conditions of stress. Mhrt binds to and sequesters a chromatin remodeling enzyme called Brg1, enabling certain key transcripts to be produced at basal levels. This projects intends use a technique called Single-Molecule Analysis of RNA Transient Structure (SiM-KARTS) to probe the structure and folding dynamics of the RNA in isolation. Small model systems based on a heart-specific lncRNA were constructed in order to provide a baseline for rigorous interpretation of SiM-KARTS data. The structure of Mhrt was also investigated using chemical probing methods. Expression of Brg1 in E. coli and insect cells was attempted to enable study of the coupling between Mhrt structure and Brg1 binding, and between Brg1 binding to Mhrt and to chromatin.

Methods
This dataset was collected through analysis of gel electrophoresis, single-molecule fluorescence imaging, and analysis of the protein content of cells.