Real-Time Condensation of Nanoconfined DNA by an Intrinsically Disordered Polycationic Protein

Abstract

The Hepatitis C viral core protein (HCVcp) is an intrinsically disordered nucleocapsid protein that induces structural transformations in the viral RNA necessary for its efficient packaging within the viral particle. The mechanism of the nucleoprotein complex (NPC) formation is mainly driven by electrostatic interactions, where HCVcp locally screens repulsion charges by acting as a macromolecular counterion (+22). In the later stage of viral assembly, the number of HCVcp molecules increases above a critical threshold concentration after which all NPCs are transformed into nucleocapsid like particles (NLP). Herein, we have used single DNA molecule nanofluidics in combination with fluorescence microscopy to monitor the interaction between HCVcp and double stranded DNA. In our novel dynamic nanofluidic device, we can confine single DNA molecules in the nanochannels and visualize their condensation under constant inflow of proteins. We observed that NPC formation incur changes in the mechanical properties of stretched DNA molecules. These changes are primarily marked by appearance of a local compaction at either end of the stretched DNA, which serves as a nucleation site for global compaction. The length of the stretched DNA molecules gradually decreases and reaches a condensed state at above threshold concentrations of HCVcp. We were also able to follow unpacking of the condensed state in real time. Proteinase K was introduced from another inlet of the dynamic device and it gradually removed the HCVcp from DNA, which in turn lead to an increase in extension of condensed DNA. Our data allowed us to investigate the formation and dissociation of the DNA:HCVcp complex in real time. On a broader level, our results highlight the use of nanofluidic channels for studying complex and dynamic DNA-protein interactions.