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

Rajhans Sharma,Sriram Kk,Erik D Holmstrom,Fredrik Westerlund

doi:10.1016/j.bbrc.2020.06.051

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.

Highlights

Single-molecule methods have revolutionized our understanding of nucleic acid-protein interactions
It is not well-known if/how hepatitis C virus core protein (HCVcp) interacts with long doublestranded DNA (dsDNA) molecules that are more similar in length to the genomic RNA (~104 nts) contained within the nucleocapsid of the hepatitis C virus
Two possible reasons for end-initiated compaction are: (i) the protein preferentially accumulates at the ends of the DNA and/or (ii) the geometry of the nanochannel favors compaction from the ends. To further explore these possibilities, we investigated the interaction between HCVcp and circular DNA, which has no well-defined ends. cDNA is notoriously difficult to study with single-molecule methods since no ends are available for chemical modifications that permit anchoring of the DNA to an immobilized substrate

Summary

Introduction

Single-molecule methods have revolutionized our understanding of nucleic acid-protein interactions. Single-molecule approaches for studying genome-length (>10 kbp) doublestranded DNA (dsDNA) include optical [1] and magnetic [2] tweezers, as well as DNA curtains [3]. One main drawback with these techniques is that they rely on anchoring at least one DNA end to a fixed substrate. This can be a substantial experimental limitation since many important biomolecular reactions occur at the ends of dsDNA (e.g., repair of double-stranded breaks and elongation of telomeric repeats). There is a strong need for single-molecule methods that allow both ends of long dsDNA molecules to be studied in real time

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Conclusion