2025_Veenbaas

This repository contains supplementary data and analysis software for the fpocketR and Frag-MaP results reported in the manuscript "RNA ligand binding pockets and where to find them".

Table of Contents

• Overview
• fpocketR
  • Analysis
  • Optimization
• Frag-MaP
  • Analysis
  • Protocol Templates
• Citation
• License

Overview

This repository provides access to the data, code, and protocols accompanying our manuscript on RNA ligand-binding pocket identification and characterization. The repository is organized around two key methodologies:

• fpocketR: A computational tool for detecting and characterizing ligand-binding pockets in RNA structures
• Frag-MaP: An experimental approach for mapping RNA-ligand interactions through chemical probing

fpocketR

fpocketR is a package for detecting, visualizing, and characterizing drug-like small-molecule binding sites in RNAs. The fpocketR software can be accessed at: https://github.com/Weeks-UNC/fpocketR

fpocketR Analysis

The fpocketR/analysis/ directory contains results and example data for various RNA systems analyzed with fpocketR:

• apo_holo (FIG 5): Analysis of 10 RNA structures in both holo (ligand-bound) and apo (ligand-free) states
• dynamic_rna (Fig 5): Application of fpocketR to RNA structures with conformational dynamics
• e_coli_ribosome (FIG S7): Analysis of pockets the E. coli ribosome (PDB: 7K00)
• group_II_intron (FIG 7): Analysis of pockets in the group II intron (PDB: 5G2X)
• training_test_sets (FIG 1, 2, 6, 8): fpocketR results (default and optimized) for the small RNA training and test sets

fpocketR Optimization

The fpocketR/optimization/ directory contains:

• code: Scripts used for parameter optimization and performance evaluation of fpocketR
• data: Raw data from the multivariate optimization process that assessed 1875 parameter combinations
• figures: Visualization of optimization results and performance metrics

Frag-MaP

Frag-MaP is a chemical probing technique that uses fully functionalized fragment probes, mutational profiling (MaP), and next-generation sequencing to identify ligand binding sites in RNA with nucleotide precision. We analyzed our Frag-Map data with the Fragmapper analysis module in RNAvigate which is available at: https://github.com/Weeks-UNC/RNAvigate

Frag-Map Analysis

The Frag-MaP/analysis/ directory includes:

• fragmap_azide_enrichment_analysis.ipynb: Jupyter notebook for analyzing Frag-MaP enrichment data
• fme3_rnavigate_samples.pkl and fme5_rnavigate_samples.pkl: Processed data files containing RNAVigate samples for each sample
• images/: Figures and visualizations derived from Frag-MaP data
• README_DATA_AVAILIBLITY.md: Information on accessing additional data (FASTQ and SHAPEMapper profiles) not included in this repository

Protocol Templates

The Frag-MaP/protocol_templates/ directory contains detailed protocols for implementing the Frag-MaP methodology:

• fragmap_probe_cells.md/pdf: Protocol for probing B. subtilis cells with fully functionalized fragment probes
• fragmap_protocol_with_enrichment.md/pdf: Enhanced Frag-MaP protocol (with azide beads) for identifying ligand binding sites in RNA

Citation

If you use the data or methodologies from this repository, please cite our paper:

S.D. Veenbaas, J.T. Koehn, P.S. Irving, N.N. Lama, & K.M. Weeks, Ligand-binding pockets in RNA and where to find them, Proc. Natl. Acad. Sci. U.S.A. 122 (17) e2422346122, https://doi.org/10.1073/pnas.2422346122 (2025).

PNAS

Accepted March 11 2025