Exploration of solid-state nanopores in characterizing reaction mixtures generated from a catalytic DNA assembly circuit.

Zhentong Zhu,Ruiping Wu,Bingling Li

doi:10.1039/c8sc04875d

Abstract

Recent advances have proven that using solid-state nanopores is a promising single molecular technique to enrich the DNA assembly signaling library. Other than using them for distinguishing structures, here we innovatively adapt solid-state nanopores for use in analyzing assembly mixtures, which is usually a tougher task for either traditional characterization techniques or nanopores themselves. A trigger induced DNA step polymerization (SP-CHA), producing three-way-DNA concatemers, is designed as a model. Through counting and integrating the translocation-induced current block when each concatemer passes through a glass conical glass nanopore, we propose an electrophoresis-gel like, but homogeneous, quantitative method that can comprehensively profile the "base-pair distribution" of SP-CHA concatemer mixtures. Due to the higher sensitivity, a number of super long concatemers that were previously difficult to detect via gel electrophoresis are also revealed. These ultra-concatemers, longer than 2 kbp, could provide a much enhanced signal-to-noise ratio for nanopores and are thus believed to be more accurate indicators for the existence of a trigger, which may be of benefit for further applications, such as molecular machines or biosensors.

Highlights

Through counting and integrating the translocation-induced current block when each concatemer passes through a glass conical glass nanopore, we propose an electrophoresis-gel like, but homogeneous, quantitative method that can comprehensively profile the “base-pair distribution” of SPCHA concatemer mixtures
Through counting and integrating the translocation-induced current blocks when each concatemer passes through a conical glass nanopore (CGN), we propose a quantitative method that can comprehensively pro le “base-pair distribution” and “trigger-speci c” signals related to step polymerization based on catalytic hairpin assembly (SP-CHA) concatemer mixtures
We report the exploration of using a solid-state nanopore to analyze an enzyme-free nucleic acid assembly, using SP-CHA as a model

Summary

Introduction

From the disclosure of the nucleic acid double helix structure[1] to the rapid development of DNA nanotechnology[2,3,4,5,6,7,8,9,10] and computing,[11,12,13,14,15,16,17,18,19,20,21,22] DNA has been engineered to be much more than genetic species, but rather to be a highly controllable “programmable material”, like LEGO bricks. The basic principle of nanopore detection is that modulations in ionic current re ect the translocation of single macromolecules through a nanoscale aperture.[38] A er more than twenty years of development and improvement, it has been proven to be a powerful analysis platform that enables label-free and separation-free single-molecule analysis.[39,40,41,42,43,44,45,46,47,48,49,50,51,52] For example, pores made of proteins (bio-pores) have shown extremely high resolution in analysing targets with small diameters.[53,54,55,56,57,58] The most representative success is the invention of the 4thgeneration of gene sequencing.[40,51,52] For comparison, the other class of pores fabricated using solid-state materials (such as silicon nitride59–65) offer several potential points of superiority over bio-pores in terms of chemical, mechanical and thermal robustness and better exibility for detecting huge target molecules, which could not pass through bio-pores. Besides conventional shortcomings, such as more difficult fabrication and poorer reproducibility, a big challenge

Methods

Results

Conclusion