DOI: 10.5061/dryad.4b8gthtnw
title: Single-molecule and bulk fluorescence and biolayer interferometry on the NAD-II riboswitch and its interactions with NMN

description: The Class II NAD+ riboswitch is a bacterial RNA that binds ligands
 containing nicotinamide. Herein, we report a fluorescence and biolayer
 interferometry study of riboswitch interactions with β-NMN. The results
 reveal a shift in the prevalence of a pseudoknot structure in the presence
 of ligand and Mg2+.
description: Bulk fluorometry, single-molecule microscopy, gel
 electrophoresis, biolayer interferometry data on the NAD-II
 riboswitch.
description: # NAD+-II riboswitch
 [https://doi.org/10.5061/dryad.4b8gthtnw](https://doi.org/10.5061/dryad.4b8gthtnw) ## Description of the data and file structure The NAD+-II riboswitch was studied using bulk and single-molecule fluorescence and biolayer interferometry. ### Files and variables #### File: NADII-data.zip **Description:**  `BLI_P1a` contains biolayer interferometry traces for P1a-modified riboswitch and fits. * "assay_full" contains times and corresponding datapoints for all samples. * 20240729_whole_align_assoc contains contains times and corresponding datapoints with the first datapoint in the association phase shifted to match for all samples. * 1to1fitassoc and 2to1fit_assoc contain 1:1 and 2:1 fits to the association phases of P1a-Cy5 RNA in 20 mM MgCl2. This data was exported from the software of a Gator Bio BLI instrument and can be opened in any program that reads text or tables. `BLI_U14` contains BLI traces and fits for U14-modified riboswitch. "assay_full" contains times and corresponding datapoints for all samples and "fits" contains 1:1 and 2:1 fits to the association phase of aminoU14 RNA in 20 mM MgCl2. `Bulk_FRET` contains six folders. Filenames indicate the sample and/or ligand measured (e.g. "buffer") followed by the ligand concentration in micromolar. * RNA, RNA_dup and RNA_trip contain emission spectra from three replicate NMN titrations of P1a-Cy5 riboswitch * mock_titration contains emission spectra from a mock titration in which buffer was added instead of NMN * buffer_scans contains emission spectra of buffer with NMN added to different concentrations * NaMN_scans contains emission spectra from a titration of P1a-Cy5 riboswitch with nicotinic acid mononucleotide `Dimer_results` contains two folders: * native_gel contains images of a native gel used for dimer analysis.  .gel files are opened in ImageJ. * smimaging contains peak locations, Cy3 and Cy5 traces, and average images from single-molecule movies used for dimer analysis. Filenames that begin with C1 come from measurements in which Cy3 and Cy5-labeled RNAs were annealed together. Filenames that begin with C3 come from measurements in which Cy3-labeled RNA was annealed and then imaged in the presence of Cy5-labeled RNA. C1_het contains these files from the illumination conditions used to observe heterodimer formation, while C1_homo contains files from the illumination conditions used to observe homodimer formation. .traces files are opened in Matlab and .pks files can be opened in any program that reads text or tables. `smFRET` contains lists of single-time FRET efficiencies that were binned together to form histograms. P1a_histograms contains data on P1a-Cy5 RNA and U14_histograms contains data on U14-Cy5 RNA. Filenames indicate the imaging condition with the number of molecules indicated by "N" (e.g. NMN_Mg_N210.dat contains data for 210 molecules recorded in the presence of NMN and Mg). .dat files can be opened in any program that reads text or tables. ## Code/software Contact [jwidom@uoregon.edu](mailto:jwidom@uoregon.edu) for custom Matlab and Mathematica code used to analyze this data.