Real-time compaction of nanoconfined DNA by an intrinsically disordered macromolecular counterion

Abstract

We demonstrate how a recently developed nanofluidic device can be used to study protein-induced compaction of genome-length DNA freely suspended in solution. The protein we use in this study is the hepatitis C virus core protein (HCVcp), which is a positively charged, intrinsically disordered protein. Using nanofluidic devices in combination with fluorescence microscopy, we observe that protein-induced compaction preferentially begins at the ends of linear DNA. This observation would be difficult to make with many other single-molecule techniques, which generally require the DNA ends to be anchored to a substrate. We also demonstrate that this protein-induced compaction is reversible and can be dynamically modulated by exposing the confined DNA molecules to solutions containing either HCVcp (to promote compaction) or Proteinase K (to disassemble the compact nucleo-protein complex). Although the natural binding partner for HCVcp is genomic viral RNA, the general biophysical principles governing protein-induced compaction of DNA are likely relevant for a broad range of nucleic acid-binding proteins and their targets.

Keywords: DNA condensation and compaction; Hepatitis C virus core protein; Intrinsically disordered protein; Macromolecular counterion; Nanofluidic device; Protein-DNA interaction.

Fig. 1.

Nanofluidic device and HCVcp. Schematics of (a) the static and (b) the dynamic nanofluidic devices, the latter of which entropically traps single DNA molecules in a specially designed (c) reaction chamber. The amino acid sequence of (d) the nucleocapsid domain of HCVcp and (e) a graphical representation of the relative abundance of each amino acid.

Fig. 2.

Real-time observation of the interaction between HCVcp and genomic-length dsDNA in a dynamic nanofluidic device. (a) Kymograph showing that HCVcp (1.0 μM) initiates compaction of T4-DNA from both ends of the molecule (at 5 fps). (b) Spatial fluorescence intensity profiles from the kymograph in (a). Kymographs showing two different sites of compaction on separate cDNA molecules (at 9 fps): either at (c) the poles (ends) of the cDNA or (d) elsewhere. For (a,c,d) the scale bars represent 5 μm for the x-axis and 5 s for the y-axis. (e) Histogram depicting the percentage of T4-DNA (N = 66) and cDNA (N = 18) molecules having compaction events that begin at either the ends (poles) of the DNA or elsewhere.

Fig. 3.

Iterative compaction of genome length dsDNA-HCVcp complexes. The compaction of YOYO-1 stained T4-DNA was induced by the addition of HCVcp (red). Disassembly of the nucleoprotein complex was induced by the addition of Proteinase K (blue). Images of (a) two extended molecules, each confined within a reaction chamber, and (b) their compact form during continuous flow of HCVcp from the shallow orthogonal slit. For (a,b) the scale bar is 5 μm. The initial compaction was not imaged to prevent photobleaching of YOYO-1. Images are used to construct (c,d) kymographs showing the subsequent disassembly of the compact nucleoprotein complexes by introducing Proteinase K into the reaction chamber from the other side of the shallow slit, which marks the beginning of the iterative cycle of dissassembly and compaction. For (c,d) the x-axis scale bar is 10 μm and the y-axis scale bar is 20 s. (e) Schematic diagram of the flow directions used to compact and disassemble the HCVcp nucleo-protein complexes.

Fig. 4.

Kymographs of cDNA cleavage in the (a) absence and (b,c) presence of HCVcp. Sketches of cDNA molecules before and after photoinduced double-stranded breaks are shown above/below each kymograph to illustrate the transition to the extended conformations. The concentration of HCVcp (red) in (b) and (c) is 1 μM and the concentration of circular DNA (black) is 5 μM base pairs. Scale bars for the x- and y-axes are 3 μm and 3 s, respectively. Histograms depicting the distribution of extensions for individual (e) λ-DNA or (f) T7-DNA molecules at different concentrations of HCVcp, where the dsDNA concentration is 5 μM base pairs. The arrows point out circular complexes and complexes consisting of two λ-DNA molecules, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)