Toehold-mediated DNA Strand Displacement Reactions Research Articles

CRISPR/Cas12a triggered SERS and naked eye dual-mode biosensor for ultrasensitive and on-site detection of nucleic acid via cascade signal amplification

Highly sensitive and on-site detection of nucleic acids has always been a critical issue in the field of analytical chemistry. Surface-enhanced Raman scattering (SERS)-based biosensing exhibits huge potential in nucleic acid detection, while the applicability is restricted in trace nucleic acid screening due to the lack of appropriate signal recognition, transducing and amplification technologies. Inspired by the specific recognition of CRISPR/Cas12a and the improved sensitivity through cascade signal amplification, we innovatively proposed a CRISPR/Cas12a triggered SERS and naked eye dual-mode biosensor for ultrasensitive and on-site detection of nucleic acid via cascade signal amplification. Upon the target DNA recognition, the activated CRISPR/Cas12a indiscriminately cleaved substrate ssDNA, leading to the failure of toehold-mediated DNA-strand displacement reaction (TSDR), and triggering hybridization chain reaction (HCR) to assemble numerous G-quadruplex/hemin DNAzyme (GQH DNAzyme) for cascade signal amplification. The generated GQH DNAzyme catalyzed the oxidation of l-cysteine to cystine, perturbing the aggregation of 4-NTP@AuNPs, resulting in significant Raman signal change. On the other hand, GQH DNAzyme catalyzed the oxidation of 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), leading to obvious color change to realize portable naked-eye detection. Through this strategy, target nucleic acid concentration was tactfully transformed into sensitive Raman and portable visualization signals, and the limit of detection were as low as 34.9 aM and 1 pM, respectively. Then, this biosensor was successfully applied to meat adulteration detection, which showed superb selectivity, sensitivity and applicability for on-site detection of trace nucleic acid in complicated food matrix.

Self-Assembly of Designed Peptides with DNA to Accelerate the DNA Strand Displacement Process for Dynamic Regulation of DNAzymes.

Toehold-mediated DNA strand displacement (TMSD) is a powerful tool for controlling DNA-based molecular reactions and devices. However, the slow kinetics of TMSD reactions often limit their efficiency and practical applications. Inspired by the chemical structures of natural DNA-operating enzymes (e.g., helicase), we designed lysine-rich peptides to self-assemble with DNA-based systems. Our approach allows for accelerating the TMSD reactions, even during multiple displacement events, enhancing their overall efficiency and utility. We found that the acceleration is dependent on the peptide's sequence, length, and concentration as well as the length of the DNA toehold domain. Molecular dynamics simulations revealed that the peptides promote toehold binding between the double-stranded target and the single-stranded invader, thereby facilitating strand displacement. Furthermore, we integrated our approach into a horseradish peroxidase-mimicking DNAzyme, enabling the dynamic modulation of enzymatic functions on and off. We anticipate that the established acceleration of strand displacement reactions and the modulation of enzymatic activities offer enhanced functionality and control in the design of programmable DNA-based nanodevices.

DNA-Based Signaling Networks for Transient Colloidal Co-Assemblies.

Programmable chemical circuits inspired by signaling networks in living cells are a promising approach for the development of adaptive and autonomous self-assembling molecular systems and material functions. Progress has been made at the molecular level, but connecting molecular control circuits to self-assembling larger elements such as colloids that enable real-space studies and access to functional materials is sparse and can suffer from kinetic traps, flocculation, or difficult system integration protocols. Herein, we report a toehold-mediated DNA strand displacement reaction network capable of autonomously directing two different microgels into transient and self-regulating co-assemblies. The microgels are functionalized with DNA and become elemental components of the network. The flexibility of the circuit design allows the installation of delay phases or accelerators by chaining additional circuit modules upstream or downstream of the core circuit. The design provides an adaptable and robust route to regulate other building blocks for advanced biomimetic functions.

Front Cover: Enzyme‐Free Nucleic Acid Circuits for Fold‐Change Detection (ChemPlusChem 9/2023)

The cover picture shows an enzyme-free nucleic acid synthetic circuit constructed by the toehold-mediated DNA strand displacement reaction, where a fold-change input (top left) elicits a similar response (bottom right). This work is anticipated to shed new light on the design of DNA dynamic circuits. More information can be found in the Research Article by Hao Pei, Tong Zhu, and co-workers.

Enzyme-Free Nucleic Acid Circuits for Fold-Change Detection.

Fold-change detection is widespread in sensory systems of various organisms. Dynamic DNA nanotechnology provides an important toolbox for reproducing structures and responses of cellular circuits. In this work, we construct an enzyme-free nucleic acid circuit based on the incoherent feed-forward loop using toehold-mediated DNA strand displacement reactions and explore its dynamic behaviors. The mathematical model based on ordinary differential equations is used to evaluate the parameter regime required for fold-change detection. After selecting appropriate parameters, the constructed synthetic circuit exhibits approximate fold-change detection for multiple rounds of inputs with different initial concentrations. This work is anticipated to shed new light on the design of DNA dynamic circuits in the enzyme-free environment.

In situ reduction of gold nanoparticles-decorated MXenes-based electrochemical sensing platform for KRAS gene detection.

In this work, gold nanoparticles@Ti3C2 MXenes nanocomposites with excellent properties were combined with toehold-mediated DNA strand displacement reaction to construct an electrochemical circulating tumor DNA biosensor. The gold nanoparticles were synthesized in situ on the surface of Ti3C2 MXenes as a reducing and stabilizing agent. The good electrical conductivity of the gold nanoparticles@Ti3C2 MXenes composite and the nucleic acid amplification strategy of enzyme-free toehold-mediated DNA strand displacement reaction can be used to efficiently and specifically detect the non-small cell cancer biomarker circulating tumor DNA KRAS gene. The biosensor has a linear detection range of 10fM -10nM and a detection limit of 0.38fM, and also efficiently distinguishes single base mismatched DNA sequences. The biosensor has been successfully used for the sensitive detection of KRAS gene G12D, which has excellent potential for clinical analysis and provides a new idea for the preparation of novel MXenes-based two-dimensional composites and their application in electrochemical DNA biosensors.

Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions.

Here, we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand displacement reactions (SDRs). The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables SDR. The kinetics of the blocker enzymatic degradation thus controls the time at which the SDR starts. By varying the concentration of the blocker strand and the concentration of the enzyme, we show that we can finely tune and modulate the delayed onset of SDR. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed SDRs using RNase H and two DNA repair enzymes (formamidopyrimidine DNA glycosylase and uracil-DNA glycosylase) and corresponding blockers. The achieved temporal delay can be programed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show three possible applications of the delayed SDRs to temporally control the ligand release from a DNA nanodevice, the inhibition of a target protein by a DNA aptamer, and the output signal generated by a DNA logic circuit.

CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.

CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.

Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a

CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.

Single-Molecule Resettable DNA Computing via Magnetic Tweezers.

DNA-based Boolean logic computing has emerged as a leading technique in biosensing, diagnosis, and therapeutics. Due to the development of the biological and chemical methods, especially the toehold-mediated DNA strand displacement (TMSD) reaction, different logic gates as well as circuits can be performed. However, most of these methods have been conducted at the bulk level, which may lead to missing information and be less controllable. Herein, we engineered single-molecule DNA computing controlled by stretching forces using magnetic tweezers. By tracking the real-time signals of the DNA extension, the output can be determined at a single base-pair resolution. A kinetics-controllable TMSD reaction was realized in the range of a ∼19-fold change of the reaction rate by different stretching forces. OR, AND, and NOT gates were also achieved. In addition, resettable DNA computing using force stretching cycles has been further exemplified. Overall, such a real-time, label-free, and force-controlled single-molecule DNA computing system provided new insight into molecular computing.

How fluorescent labels affect the kinetics of the toehold-mediated DNA strand displacement reaction

End modification of the toehold domain or near to it using fluorophore dyes or quenchers can significantly modulate the kinetics of the toehold-mediated strand displacement reaction (TMSDR) by almost two orders of magnitude.

Programming cell entry of molecules via reversible synthetic DNA circuits on cell membrane

Cellular uptake of biomolecules is crucial for regulating cell function. However, powerful and biocompatible tools for dynamically manipulating the cell entry of single-stranded DNAs (ssDNAs) remain elusive. Herein, we constructed synthetic DNA circuits on the cell membrane to program the cell entry of ssDNAs, using toehold-mediated DNA strand displacement reactions. We found that the dimerization and trimerization of cholesterol-ssDNAs enhanced membrane-anchoring and cellular uptake of ssDNAs. Moreover, we demonstrated that de-dimerization and de-trimerization of cholesterol-ssDNAs could be accomplished by inputting recovery ssDNAs into the synthetic DNA circuits, which could simultaneously decrease the cellular uptake of ssDNAs. We speculate that operating the synthetic DNA circuits on the cell membrane will be a powerful strategy for regulating the cellular uptake of exogenous materials, which has important implications for bioimaging, drug delivery, and gene therapy.

Bioinspired Programmable Engineering of a Color-Change Biointerface based on Dual-Stimulation Regulation.

Some animals can change their skin colors in response to multiple stimuli by controlling the skin pigment cell or guanine crystals. An artificial color-change interface inspired by these natural properties has been developed; the interface responded to different types of stimuli, however, is still rare. Herein, we design a color-change biointerface that responds to the cooperative stimulation of light and DNA by changing the conformation of DNA structures to achieve programmable activation of the interface. We construct the biointerface by the combination of a fluorescent dye-labeled hairpin DNA with a photoresponsive molecule, which is cleaved by the first stimulation of UV light (stimulus 1, S1) to expose the toehold domain. Then, the biointerface gets color changed through the toehold-mediated DNA strand displacement reaction with the second stimulation of the invading DNA probe (stimulus 2, S2). Moreover, the transformation efficiency of the biointerface increased from 6.8 to 64% with an increase of S1 from 0 to 600 s in the presence of S2. Also, after the stimulation of S1, the effective transformation efficiency was also tuned from 5.3 to 72% by different amounts of S2 in the complex matrix, indicating the potential for a high response in real-world settings.

Label-free and self-assembled fluorescent DNA nanopompom for determination of miRNA-21.

A label-free fluorescence method based on self-assembled DNA nanopompom has been developed for miRNA-21 detection. In the presence of miRNA-21, three DNA hairpin probes with split G-quadruplex assemble the DNA nanopompom. Based on the isothermal toehold-mediated DNA strand displacement reaction, the target miRNA can be catalytically recycled and trigger three DNA hairpin probes to self-assemble the DNA nanopompom and release the G-quadruplex. The formation of the G-quadruplex increases the fluorescence emission intensity of thioflavin. For thioflavin-based miRNA-21 detection, the excitation and emissionwavelengths are set to425nm and 490 nm, respectively. The limit of detection for miRNA-21 is 0.8pM according to F/F0 = 0.0031 × CmiRNA-21 + 1.0382 (R2 = 0.9978). This sensing system provides a low-cost, effective, and convenient method for miRNA detection, which holds great potential in biochemical diagnosis and clinical practice. Graphical abstract Label-free and self-assembled fluorescent DNA nanopompom for miRNA detection.

Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated DNA Strand Displacement Reaction on Magnetic Beads: Toward Ultrasensitive Energy-Transfer-Based Photoelectrochemical Detection of miRNA-141 in Real Blood Sample.

Highly stable circulating microRNAs (miRNAs) are currently recognized as a novel potential biomarker for clinical cancer diagnosis in the early stage. However, limited by its low concentration, high sequence similarity, as well as the numerous interferences in body fluids, detection of miRNA in whole blood with sufficient selectivity and sensitivity is still challenging. Herein, we reported the integration of entropy-driven toehold-mediated DNA strand displacement (ETSD) reaction with magnetic beads (MB) toward the energy-transfer-based photoelectrochemical (PEC) detection of the prostate carcinoma (PCa) biomarker miRNA-141 in a real blood sample. In this protocol, the ETSD reaction was divided into two steps, and cooperated with magnetic separation, target extraction and amplification could be realized in a single test and ultrasensitive detection of miRNA-141 could be achieved in undiluted whole blood sample. This work proposed a new solution for sensitive biomolecular detection in a complex biological milieu and exhibited great promise for future clinical cancer diagnosis.

Flexible regulation of DNA displacement reaction through nucleic acid-recognition enzyme and its application in keypad lock system and biosensing

Toehold-mediated DNA strand displacement reaction (SDR) plays pivotal roles for the construction of diverse dynamic DNA nanodevices. To date, many elements have been introduced into SDR system to achieve controllable activation and fine regulation. However, as the most relevant stimuli for nucleic acid involved reaction, nucleic acid-recognizing enzymes (NAEs) have received nearly no attention so far despite SDR often takes place in NAEs-enriched environment (i.e., biological fluids). Herein, we report a set of NAEs-controlled SDR strategies, which take full advantage of NAEs’ properties. In this study, three different kinds of enzymes belonging to several classes (i.e., exonuclease, endonuclease and polymerase) have been used to activate or inhibit SDR, and more importantly, some mechanisms behind these strategies on how NAEs affect SDR have also been revealed. The exploration to use NAEs as possible cues to operate SDR will expand the available toolbox to build novel stimuli-fueled DNA nanodevices and could open the door to many applications including enzyme-triggered biocomputing and biosensing.

DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes.

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.

DNA photonic nanowires with tunable FRET signals on the basis of toehold-mediated DNA strand displacement reactions.

A DNA photonic nanowire with tunable FRET signals was fabricated on the basis of cascaded toehold-mediated DNA strand displacement reactions. Different DNA inputs were added to trigger the reaction network, and the corresponding FRET signals were obtained. Compared to the direct hybridization, this design is sensitive for 2 nM targets within 20 min and also causes color changes of the solution with blue-light excitation. It could also be applied in live cells to monitor MicroRNA with a simple modification which might become a low-cost method for further application in the future.

A fluorescence biosensor for VEGF detection based on DNA assembly structure switching and isothermal amplification

Vascular endothelial growth factor (VEGF) is an important biomarker in cancer angiogenesis. Here, we develop a aptasensor method for VEGF detection based on DNA assembly structure switching and isothermal amplification. The design employs a DNA assembly made of a isothermal amplification template, a aptamer, a primer and a protector chain. The DNA assembly is unable to undergo isothermal amplification in the absence of target. The presence of the target, however, triggers a structure switching event that causes hybridization of primer with template to facilitate isothermal amplification. Whereafter, toehold-mediated DNA strand displacement reaction between the generated (single-stranded DNA) ssDNA and fluorescent/quencher labeled probe are performed. Then, the increase in fluorescence provides an analytical signal. This strategy opens up a sensitive, selective and simple sensing platform for detection of VEGF. The system was also implemented to analyze the VEGF in human serum samples with satisfactory results.

DNA and DNA computation based on toehold-mediated strand-displacement reactions

biocompatibility. Considering the critical role of DNA less than 150 base pairs (bp) in cellular processes such as regulated gene expression, quantifying the intrinsic bend ability of DNA on a sub-persistence length scale is essential to understanding its molecular functions and the DNA-protein interaction. From the classical point of view, double-stranded DNA is assumed to be stiff and can be treated by semi-flexible chain, but recent studies have yielded contradictory results. A lot of studies tried to prove that the worm-like chain model can be used to fully describe DNA chain. However, recent theoretical and experimental studies indicated that DNA exhibits high flexibility on a short length scale, which cannot be described by the worm-like chain model. Further studies are needed to address the extreme flexibility of DNA on a short length scale. On the basis of the predictability of the double helical structure and the Watson-Crick binding thermodynamics for DNA, a class of DNA reactions can be defined, called toehold-mediated strand-displacement reaction, in which one complementary single-stranded DNA sequence first binds to the dangling toehold domain of the substrate in a pre-hybridized double-stranded DNA, then triggers the strand-displacement reaction, and finally results in the dissociation of the third strand previously bound to the substrate with partial complementarity. In dynamic DNA nanotechnology, isothermal toehold-mediated DNA strand-displacement reaction has been used to design complex nanostructure and nanodevice for molecular computation. The kinetics of the strand-displacement can be modulated using the toehold length. In order to weaken the coupling between the kinetics of strand-displacement and the thermodynamics of the reaction, the concept of toehold exchange was introduced by Winfree et al. to improve the control of strand-displacement kinetics. More importantly, the biomolecular reaction (BM) rate constant of toehold exchange can be analytically derived using the three-step model. Through utilizing strand-displacement reactions and taking advantage of its programmable sequences and precise recognition properties, DNA can be used to build complex circuits which can proceed robustly at constant temperature, achieving specific functions. DNA strand-displacement reaction can be employed to fabricate logic gates, and large and complex circuits for DNA computing, to mimic the naturally occurring occurrence of biological systems. Based on that, DNA circuit can then be used to direct the assembly of nanodevice following the designed pathway, and modulate the chemical reaction networks on the surface of living cell or in cellular systems for biosensing, even program the cellular machinery in the future for genetic diagnostic or gene therapy. In the present paper, we reviewed the proceedings in the fields of DNA structure and conformational changes, and DNA flexibility, discussed the mechanism of DNA strand-displacement reaction at the molecular level, and introduced the recent studies in DNA computation as well as the dynamic DNA nanotechnology, such as self-assembly.