DNA toroids form via a flower intermediate

TL;DR

This study uncovers a detailed pathway for DNA toroid formation involving a flower intermediate, combining AFM imaging and dynamic folding assays to reveal the stepwise condensation process mediated by protamine.

Contribution

It introduces a nucleation-growth model for DNA toroid formation that includes a flower-shaped intermediate, advancing understanding of DNA condensation mechanisms.

Findings

DNA folds into a flower structure at low protamine concentrations.

Initial folding involves multiple loops with similar dynamics to protamine bending.

Higher protamine concentrations produce small, vertically packed toroids.

Abstract

DNA in sperm cells must undergo an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is condensed by protamine, a positively charged protein that loops the DNA into a toroid. Our goal is to determine the pathway and mechanism for toroid formation. We first imaged short-length (L=217-1023 nm) DNA molecules in 0-5.0 $\mu$M protamine using an atomic force microscope (AFM). At low protamine concentrations (0.2-0.6 $\mu$M), molecules dramatically condensed, folding into a flower structure. Dynamic folding measurements of the DNA using a tethered particle motion (TPM) assay revealed a corresponding, initial folding event, which was >3 loops at L=398 nm. The initial folding event was made up of smaller (<1 loop) events that had similar dynamics as protamine-induced bending. This suggests that flowers form in an initial step as protamine binds and bends the DNA. It was not until higher protamine concentrations (>2 $\mu$M) that DNA in the AFM assay formed small (<10 loop), vertically packed toroids. Taken together, these results lead us to propose a nucleation-growth model of toroid formation that includes a flower intermediate. This pathway is important in both in vivo DNA condensation and in vitro engineering of DNA nanostructures.