DNA toroids form via a flower intermediate

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 $μ$M protamine using an atomic force microscope (AFM). At low protamine concentrations (0.2-0.6 $μ$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 $μ$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.

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