Complex DNA knots detected with a nanopore sensor

Rajesh Kumar Sharma,Ishita Agrawal,Patrick S Doyle,Slaven Garaj,Liang Dai

doi:10.1038/s41467-019-12358-4

Abstract

Equilibrium knots are common in biological polymers—their prevalence, size distribution, structure, and dynamics have been extensively studied, with implications to fundamental biological processes and DNA sequencing technologies. Nanopore microscopy is a high-throughput single-molecule technique capable of detecting the shape of biopolymers, including DNA knots. Here we demonstrate nanopore sensors that map the equilibrium structure of DNA knots, without spurious knot tightening and sliding. We show the occurrence of both tight and loose knots, reconciling previous contradictory results from different experimental techniques. We evidence the occurrence of two quantitatively different modes of knot translocation through the nanopores, involving very different tension forces. With large statistics, we explore the complex knots and, for the first time, reveal the existence of rare composite knots. We use parametrized complexity, in concert with simulations, to test the theoretical assumptions of the models, further asserting the relevance of nanopores in future investigation of knots.

Highlights

Equilibrium knots are common in biological polymers—their prevalence, size distribution, structure, and dynamics have been extensively studied, with implications to fundamental biological processes and DNA sequencing technologies
We demonstrated the use of nanopores for investigating the complex conformation space of DNA knots
While discussing the inherent limitations of nanopore-based detection of knots, we show how statistical analysis can be implemented to overcome these limitations and gain relevant insights about the complex knotting and knot translocation phenomena

Summary

Introduction

Equilibrium knots are common in biological polymers—their prevalence, size distribution, structure, and dynamics have been extensively studied, with implications to fundamental biological processes and DNA sequencing technologies. We demonstrate that the nanopore sensor optimally designed, could map the equilibrium configuration of the DNA knots, without nanopore-induced sliding or tightening of the knots We use such nanopores to explore the distribution and complexity of the knots in DNA molecules with unprecedented statistics, and compare the results with numerical simulations. While bulk measurements offer only ensemble-averaged properties of the knots without intricate information, the single-molecule experimental techniques—such as optical tweezers[31], fluorescence imaging in micro-fluidic and nano-fluidic channels[32,33,34], and electron and atomic force microscopies35–37—are limited to the investigation of small molecules and low statistics To bridge this statistical gap, we employ nanopore microscopy, as a singlemolecule technique with a comparatively high throughput, which allows us to investigate the dominant configurations, and the infrequent events. The magnitude of the blocked current is a very sensitive measure of the geometrical and physical properties of the part of the biomolecule residing within the sensing region of the nanopore at that given instance

Methods

Results

Conclusion