Single Molecular Nanopores as a Label-Free Method for Homogeneous Conformation Investigation and Anti-Interference Molecular Analysis

Abstract

In this paper, we propose a “reciprocal strategy” that, on the one hand, explores the ability of solid-state nanopores in a homogeneous high-fidelity characterization of nucleic acid assembly and, on the other hand, the formed nucleic acid assembly with a large size serves as an amplifier to provide a highly distinguished and anti-interference signal for molecular sensing. Four-hairpin hybridization chain reaction (HCR) with G-rich tail tags is taken as the proof-of-concept demonstration. G-rich tail tags are commonly used to form G-quadruplex signal probes on the side chain of HCR duplex concatemers. When such G-tailed HCR concatemers translocate the nanopore, abnormal, much higher nanopore signals over normal duplexes can be observed. Combined with atomic force microscopy, we reveal the G-rich tail may easily induce the “intermolecular interaction” between HCR concatemers to form “branched assembly structure (BAS)”. To the best of our knowledge, this is the first evidence for the formation BAS of the G tailed HCR concatemers in a homogeneous solution. Systematic nanopore measurements further suggest the formation of these BASs is closely related to the types of salt ions, the amount of G, the concentration of substrate hairpins, the reaction time, and so forth. Under optimized conditions, these BASs can be grown to just the right size without being too large to block the pores, while producing a current 14 times that of conventional double-stranded chains. Here, these very abnormal large current blockages have, in turn, been taken as an anti-interference signal indicator for small targets in order to defend the high noises resulting from co-existing big species (e.g., enzymes or other long double-stranded DNA).

Solid-State Nanopore Sensors with Enhanced Sensitivity through Nucleic Acid Amplification

“…HCR can be triggered specifically by initiator, ultimately forming double-stranded DNA (Figure 5a). 62 In order to analyze the structure of HCR products, two "tailing designs" were selected (Figure 5b). The tail design is a complete Gquadruplex, and the tail design is split mode (Figure 5c).…”

“…HCR can be triggered specifically by initiator, ultimately forming double-stranded DNA (Figure a) . In order to analyze the structure of HCR products, two “tailing designs” were selected (Figure b).…”

Nanoconfined Electrokinetic Chromatography (NEC): Gradient Separation and Sensing of Short DNA Fragments at the Single-Molecule Level

“…Despite the high reproducibility and stableness of biological nanopores, − solid-state nanopores are attractive due to their higher robustness and stability . Solid nanopores offer adjustable pore size with diameters ranging from several nanometers to several hundreds of nanometers during the fabrication process, allowing for the detection of a wide range of analytes. − Resistive pulse sensing with nanopores has recently gained significant attention as an appealing method to study DNA fragments. − Nanopores have proved successful in detecting diverse DNA nanostructures, including DNA tetrahedrons, , multiarm DNA concatemers, , concentric square DNA origami, , and many more.…”

A novel design of DNA duplex containing programmable sensing sites for nanopore-based length-resolution reading and applications for Pb2+ and cfDNA analysis

“…Studies involving nucleic acid tetrahedrons and nucleic acid cubes as signal transducers 15,16 or using loop-mediated isothermal amplification (LAMP), 17 hybridization chain reaction (HCR), 18 and CRISPR-Cas technologies as amplification methods 19,20 have been proposed. These methods are known as indirect detection methods.…”