Catalytic Hairpin Assembly‐Programmed DNA Three‐Way Junction for Enzyme‐Free and Amplified Electrochemical Detection of Target DNA

Abstract

DNA three-way junctions (DNA 3WJ) have been widely used as important building blocks for the construction of DNA architectures and dynamic assemblies. Herein, we describe for the first time a catalytic hairpin assembly-programmed DNA three-way junction (CHA-3WJ) strategy for the enzyme-free and amplified electrochemical detection of target DNA. It takes full advantage of the target-catalyzed hairpin assembly-induced proximity effect of toehold and branch-migration domains for the ingenious execution of the strand displacement reaction to form the DNA 3WJ on the electrode surface. A low detection limit of 0.5 pM with an excellent selectivity was achieved for target DNA detection. The developed CHA-3WJ strategy also offers distinct advantages of simplicity in probe design and biosensor fabrication, as well as enzyme-free operation. Thus, it opens a promising avenue for applications in bioanalysis, design of DNA-responsive devices, and dynamic DNA assemblies.

Principles and Applications of Nucleic Acid Strand Displacement Reactions

“…On the basis of catalytic hairpin assembly, a wide range of sensors has been developed over the past few years. With the use of similar principles as the HCR sensors discussed above, the CHA reaction has been applied to the detection of the same analyte classes (i.e., metal ions, − DNA and DNA mismatches, − mRNA, microRNAs, − ,− proteins, ,− and cells) . As for the HCR, analytes other than nucleic acids can be detected by coupling the CHA scheme to aptamer target recognition.…”

Self-assembled DNA dendrons as signal amplifiers in a DNA probe-based chemiluminescence assay for enhanced colorimetric detection of short target cDNA

“…Notably, the DNA nanostructures are sufficiently produced even with a small amount of target DNA, with all hairpin DNA being completely consumed in the target DNA recycling CHA reactions. [13][14][15][16] The resultant DNA nanostructures in combination with various detection indicators including fluo-rescence resonance energy transfer (FRET), [17][18][19] chemiluminescence (CL), [20][21][22] and electrochemistry [23][24][25] effectively detect short target DNA with high sensitivity and specificity.…”

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

“…Some of the challenges demonstrated by a simple stem‐loop probe for electrochemical nucleic acid detection were overcome in the case of the three‐way junction (3WJ) . This design allowed the response to increase with the target concentration (“OFF‐ON” signal), since it utilized an additional redox marker‐containing strand, which was complementary to both the stem‐loop probe and the target.…”

“…This design allowed the response to increase with the target concentration (“OFF‐ON” signal), since it utilized an additional redox marker‐containing strand, which was complementary to both the stem‐loop probe and the target. Thus, the target triggered formation of the 3WJ structure with the stem‐loop and additional strand, in which the redox marker was brought close to the electrode's surface, resulting in a current density signal proportional to the target concentration (Scheme B) . However, the selectivity of the 3WJ design still did not allow discrimination of SNS, since it was still stable when an unpaired nucleotide was present .…”

“…Although this sensor design is widely used, it presents a number of significant limitations. First, stem‐loop folded probes have limited selectivity, since they do not differentiate single nucleotide substitutions (SNS) at room temperatures (∼22 °C) . Second, being folded themselves, such probes are unable to efficiently interrogate single‐stranded DNA or RNA folded in stable secondary structures .…”