Data from: Protamine loops DNA in multiple steps
Data files
Mar 24, 2026 version files 11.63 MB
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Library_of_Singlets.zip
11.63 MB
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README.md
3.37 KB
Abstract
Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius, and some of the folded molecules are partial loops—c-shapes or s-shapes—that have a radius of curvature of ∼10 nm. Further analysis of these structures suggests that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.
Dataset DOI: 10.5061/dryad.ngf1vhj8d
Description of the data and file structure
AFM assay data collection and analysis
We used a Dimension 3000 AFM (Digital Instruments) with a Nanoscope IIIa controller to image samples and PPP-XYNCSTR-model cantilevers (NanoSensors, resonant frequency = 150 kHz, force constant = 7.4 N/m, length = 150 μm, tip radius < 7 nm) (Supplementary Figure S1A). All images were taken in tapping mode. The scan rate ranged from 1 to 6 Hz. Images are 1 μm × 1 μm and contain 256 × 256 pixels. Images were processed using Gwyddion (36) or SPIP. For all images, we aligned rows using a fifth-degree polynomial.
To create a library of images of single DNA molecules (Supplementary Table S1), we selected single molecules using ImageJ v1.51 (37,38) that were at least 1 pixel away from other molecules and were contained within the field of view (Supplementary Figure S1B). To be selected, molecules also had to lay flat along the surface (having a contour length of 80% of the actual value) and have the expected height of ∼0.5 nm. We cropped a 200 nm × 200 nm image and thresholded it so that the DNA was white against a black background.
Files and variables
File: Library_of_Singlets.zip
Description:
This library contains the 200 nm x 200 nm images of each of the singlet DNA molecules and the thresholded images in the "BW" folders. The singlets are arranged into folders in the file structure by DNA length (50, 105, and 217 nm) and protamine concentration (0, 0.2, 0.3, 0.6, and 2 microMolar).
Code/software
Files are in .jpg format.