Detecting topological variations of DNA at single-molecule level

Ke Liu,Chao Pan,Alexandre Kuhn,Georg E Fantner,Adrian Pascal Nievergelt,Olgica Milenkovic,Aleksandra Radenovic

doi:10.1038/s41467-018-07924-1

Abstract

In addition to their use in DNA sequencing, ultrathin nanopore membranes have potential applications in detecting topological variations in deoxyribonucleic acid (DNA). This is due to the fact that when topologically edited DNA molecules, driven by electrophoretic forces, translocate through a narrow orifice, transient residings of edited segments inside the orifice modulate the ionic flow. Here we utilize two programmable barcoding methods based on base-pairing, namely forming a gap in dsDNA and creating protrusion sites in ssDNA for generating a hybrid DNA complex. We integrate a discriminative noise analysis for ds and ss DNA topologies into the threshold detection, resulting in improved multi-level signal detection and consequent extraction of reliable information about topological variations. Moreover, the positional information of the barcode along the template sequence can be determined unambiguously. All methods may be further modified to detect nicks in DNA, and thereby detect DNA damage and repair sites.

Highlights

In addition to their use in DNA sequencing, ultrathin nanopore membranes have potential applications in detecting topological variations in deoxyribonucleic acid (DNA)
The two types of nanopores differ in several aspects: First, biological pores have well-defined pore geometries with prescribed atomic precision
Biological pores have some intrinsic drawbacks: The fragility of the lipid bilayer that supports the pore; the technical difficulties encountered to graft biological pores into a large-scale array; and most importantly, the structural drawback related to the long β-barrel in α-hemolysin which “dilutes” the ionic current signal of a single nucleotide from the narrow constriction

Summary

Introduction

In addition to their use in DNA sequencing, ultrathin nanopore membranes have potential applications in detecting topological variations in deoxyribonucleic acid (DNA). The lipid bilayer used to host biological pores can be chosen to have a low dielectric constant for low-noise sensing Despite these advantageous features, biological pores have some intrinsic drawbacks: The fragility of the lipid bilayer that supports the pore; the technical difficulties encountered to graft biological pores into a large-scale array (more than 1,000,000 pores/cm2); and most importantly, the structural drawback related to the long β-barrel in α-hemolysin which “dilutes” the ionic current signal of a single nucleotide from the narrow constriction. Bell et al.[24] exploited a digital encoding method to study the long-range arrangement of targeted sites along DNA strands As a result, this raises the issue of the uncertainty in resolving the localized information of a feature along the DNA strand. The hypothetical current trace with two discrete levels captures the structure of our complex (Fig. 1f)

Methods

Findings

Conclusion