Single-molecule microscopy (SiM-KARTS) and chemical probing (SHAPE) of lncRNA
Data files
Jul 29, 2025 version files 431.58 MB
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AHA_Data_Deposit_2025.zip
431.57 MB
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README.md
1.77 KB
Abstract
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.
Dataset DOI: https://doi.org/10.5061/dryad.j9kd51cnp
Description of the data and file structure
AHA_Data_Deposit_2025.zip contains data collected for three experimental efforts, separated into sub-folders:
- Single-molecule fluorescence microscopy on a segment of the long noncoding RNA* Braveheart*
- Chemical secondary structure probing of long noncoding RNA* Myheart*
- Expression of Brg1 protein in SF9 insect cells
Files and variables
For convenience, each sub-folder contains a PDF describing the file naming conventions, data structure and units of measurement of the files within it. The folders are:
1_single-molecule/Dwell time files (dwell times, in movie frames, in unbound and probe-bound states of the RNA)
1_single-molecule/Movie files (single-molecule fluorescence images and traces)
2_gels (polyacrylamide gel electrophoresis images)
3_protein-expression/Data Images (images of bacterial plates and SDS-PAGE gels from attempted preparation of Brg1 protein)
3_protein-expression/Gene Sequencing Data (Sanger sequencing data demonstrating cloning of Brg1 gene into plasmids)
Code/software
Most files can be opened using free software, as detailed below. All versions appear to be compatible.
.tif files can be opened in any software capable of opening images
.gel files can be opened in ImageJ, Fiji, or Amersham Typhoon imaging software
.dat and .pks files can be opened in any software capable of reading text files
.traces files are opened in Matlab. Code to open .traces files and convert them to simple tables (including instructions) is provided in the folder “1_single-molecule/Movie files”.
This dataset was collected through analysis of gel electrophoresis, single-molecule fluorescence imaging, and analysis of the protein content of cells.