Probing Spatial Organization of DNA Strands Using Enzyme-Free Hairpin Assembly Circuits

Abstract

Catalyzed hairpin assembly (CHA) is a robust enzyme-free signal-amplification reaction that has a wide range of potential applications especially in biosensing. Although most studies on the analytical applications of CHA focus on the measurement of concentrations of biomolecules, we show here CHA can also be used to probe the spatial organization of biomolecules such as single-stranded DNA. The basis of such detection is the fact that a DNA structure that brings a toehold and a branch migration domain into close proximity can catalyze the CHA reaction. We quantitatively studied this phenomenon and applied it to the detection of domain reorganization that occurs during DNA self-assembly processes such as hybridization chain reaction (HCR). We also show CHA circuits can be designed to detect certain types of hybridization defects. This principle allowed us to develop a ‘signal-on’ assay that can simultaneously respond to multiple types of mutations in a DNA strand in one simple reaction, which is of great interest in genotyping and molecular diagnostics. These findings highlight the potential impacts of DNA circuitry on DNA nanotechnology and provide new tools for further development of these fields.

Availability: A Metric for Nucleic Acid Strand Displacement Systems

“…11 Even this small leakage drastically limits the scalability of feed-forward, cross-catalytic, and autocatalytic networks, where fuel invasion will unintentionally release the catalyst of the coupled networks. Thermal fluctuations such as fraying have long been suspected as the source of intrinsic leakage, and strategies to suppress it include (1) careful sequence and domain design such as using GC pairs at the fraying locations, 24 (2) use of proper reaction conditions, 47 (3) use of GC-rich sequences or introduction of buffer or clamping domains that are absent from fuel sequences, 36,40 (4) sequestration of domains in hairpin structures, 48 (5) use of extremely pure DNA strands made in bacteria, 26 (6) incorporation of mismatches, 39 and (7) novel domain level redundancy. 49 While each of these approaches has shown some effect, a clear set of design rules have not emerged for consistently and efficiently reducing leakage.…”

A versatile immobilization-free photoelectrochemical biosensor for ultrasensitive detection of cancer biomarker based on enzyme-free cascaded quadratic amplification strategy

“…After CHA reaction, domain-m, domain-d, and domain-f in CHA-HP1 are colocalized with domain-j* and domain-g in CHA-HP2 at the same end of the CHA-HP2@CHA-HP1 duplex complex. This bound 'toehold' and 'branchmigration' segments could then open HCR-HP1 (Reaction 5) (Chen, 2012;Li et al, 2012) through strand displacement, which could further initiate the downstream HCR amplification circuit. The added two bulged thymidines (domain-e) between domain-g and domain-a in CHA-HP2 could not only accelerate this strand displacement (Chen, 2012), but also stabilize the formed threeway junctions by allowing coaxial stacking of two of the three duplexes (Leontis et al, 1991).…”

An enzyme-free and label-free fluorescent biosensor for small molecules by G-quadruplex based hybridization chain reaction

“…HCR not only provides an enzyme-free signal amplification method but also involves a unique assembly process, which works under mild conditions with no specific demand for ionic strength, pH or temperature. Nowadays, many studies combined the amplification capability of HCR with various sensing platforms and showed promising results for the detection of biomolecules such as DNA [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22], microRNA [23,24], proteins [25][26][27][28][29][30][31], cells [32,33]. However, to this day there are few reports for the detection of small molecules [34][35].…”