Effects of Fluorescent Pair on the Kinetics of DNA Strand Displacement Reaction

Fluorescent labels are widely used in the characterizations of DNA-based reaction network operations.We systematically studied the effects of commonly used fluorescent pairs on thermal stabilities of signal-substrate duplex and the strand displacement kinetics.It is demonstrated that the modifications of duplex with fluorescent pairs stabilize DNA duplex by up to 3.5°C,and the kinetics of DNA strand displacement circuit is also evidently slowed down.These results highlight the importance of fluorescent pairs towards the kinetic modulation in designing nucleic acid probes and complex DNA dynamic circuits.

Quantum Dot Nanobeads-Labelled Lateral Flow Immunoassay Strip for Rapid and Sensitive Detection of Salmonella Typhimurium Based on Strand Displacement Loop-Mediated Isothermal Amplification

Rapid,sensitive,point-of-care detection of pathogenic bacteria is important for food safety.In this study,we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip(QBs-labelled LFIAS)combined with strand displacement loop-mediated isothermal amplification(SD-LAMP)for quantitative Salmonella Typhimurium(ST)detection.Quantum dot nanobeads(QBs)served as fluorescence reporters,providing good detection efficiency.The customizable strand displacement(SD)probe was used in LAMP to improve the specificity of the method and prevent by-product capture.Detection was based on a sandwich immunoassay.A fluorescence strip reader measured the fluorescence intensity(FI)of the test(T)line and control(C)line.The linear detection range of the strip was 10^(2)–10^(8) colony forming units(CFU)·mL^(-1).The visual limit of detection was 10^(3) CFU·mL^(-1),indicating that the system was ten-fold more sensitive than AuNPs-labelled test strips.ST specificity was analyzed in accordance with agarose gel outputs of polymerase chain reaction(PCR)and SD-LAMP.We detected ST in foods with an acceptable recovery of 85%–110%.The method is rapid,simple,almost equipment-free,and suitable for bacterial detection in foods and for clinical diagnosis.

Toeless and reversible DNA strand displacement based on Hoogsteen-bond triplex

Strand displacement reaction is a crucial component in the assembly of diverse DNA-based nanodevices,with the toehold-mediated strand displacement reaction representing the prevailing strategy.However,the single-stranded Watson-Crick sticky region that serves as the trigger for strand displacement can also cause leakage reactions by introducing crosstalk in complex DNA circuits.Here,we proposed the toeless and reversible DNA strand displacement reaction based on the Hoogsteen-bond triplex,which is compatible with most of the existing DNA circuits.We demonstrated that our proposed reaction can occur at pH 5 and can be reversed at pH 9.We also observed an approximately linear relationship between the degree of reaction and pH within the range of pH 5-6,providing the potential for precise regulation of the reaction.Meanwhile,by altering the sequence orientation,we have demonstrated that our proposed reaction can be initiated or regulated through the same toeless mechanism without the requirement for protonation in low pH conditions.Based on the proposed reaction principle,we further constructed a variety of DNA nanodevices,including two types of DNA logic gates that rely on pH 5/pH 9 changes for initiating and reversing:the AND gate and the OR gate.We also successfully constructed a DNA Walker based on our proposed reaction modes,which can move along a given track after the introduction of a programmable DNA sequence and complete a cycle after 4 steps.Our findings suggest that this innovative approach will have broad utility in the development of DNA circuits,molecular sensors,and other complex biological systems.

Regulable toehold lock for the effective control of strand displacement reaction sequence and circuit leakage

Strand displacement reaction enables the construction of enzyme-free DNA reaction networks,thus has been widely applied to DNA circuit and nanotechnology.It has the characteristics of high efficiency,universality and regulatability.However,the existing regulation tools cannot enable effective control of the reaction sequence,which undoubtedly limits the construction of complex nucleic acid circuits.Herein,we developed a regulation tool,toehold lock,and achieved strict control of reaction sequence without loss of the main reaction signal output.Furthermore,we applied the tool to scenarios such as seesaw circuits,AND/OR logic gates,and entropy-driven circuits,and respectively demonstrated its significant superiority compared to the original method.We believe that the proposed toehold lock has greatly optimized the efficiency of DNA strand displacement-based networks,and we anticipate that the tool will be widely used in multiple fields.

Switching On/Off of Guide RNA by Photoinduced Strand Displacement for Functional Control of CRISPR/Cas9

Functional control of CRISPR/Cas9 is essential for precise gene manipulation.Chemical engineering of guide RNA(gRNA)provides diverse approaches for conditional control of CRISPR/Cas9 function with a variety of chemical reactive groups.However,previous investigations into chemically engineering gRNA only unidirectionally regulated the CRISPR/Cas9 function via stimuli-induced caging/decaging processes.Herein,we propose a combinatory strategy to engineer the dynamics of gRNA in which photocontrolled strand-displacement reactions coupled with sequence designs of gRNA can achieve lightinduced switching-on/off control of CRISPR/Cas9 function.Biochemical analysis and cellular gene regulation indicate this approach is capable of both activating and deactivating CRISPR/Cas9 activities using light irradiation.Moreover,photocontrolled multiplex modulations of gene expression for opposite regulatory effects have also been achieved simultaneously under the same cellular context.This work establishes an essential principle for construction of stimuli-induced switching-on/off modulations of gRNA that can greatly enrich the versatility of conditional control for a variety of CRISPR/Cas9-based applications.

Visually Intracellular Detection of Telomerase Activity Based on DNA Strand Displacement Reaction and Gold Nanoparticle Labeling

Telomerase,which is regarded as a common biomarker for early cancer diagnostics and a potential target for clinical therapies,has attracted considerable interests concerning its detection and monitoring.Herein,we propose a sensitive method by designing a gold nanoparticle(AuNP)probe for visually intracellular detection of telomerase activity.The AuNPs were functionalized with a telomer-ase substrate primer(SH-prime).A 6-carboxy-fluorescein(FAM)modified strand(FAM-probe)was attached to the surface of AuNP through its complementary stand(SH-attach).In the absence of telomerase,the fluorescence resonance energy transfer(FRET)from FAM to AuNPs results in efficient fluorescence quenching.In the presence of telomerase,SH-primers on AuNPs were extended with the repeat units(TTAGGGG)n.The extension sequence triggered the strand displacement of FAM-probe to restore the fluorescence signals.It is worth mentioning that the proposed strategy does not need to design complex hairpin structure and allows the meas-urement of telomerase in crude cell extracts down to 0.5 HeLa cells/μL in 2 h.In addition,the present sensing platform can be ap-plied to the visually intracellular detection of telomerase activity in living cells.

Development of DNA computing and information processing based on DNA-strand displacement

DNA computing, currently a hot research field in information processing, has the advantages of parallelism, low energy consumption, and high storability, therefore, it has been applied to a variety of complicated computational problems. The emerging field of DNA nanotechnology has also developed quickly; within it, the method of DNA strand displacement has drawn great attention because it is self-induced, sensitive, accurate, and operationally simple. This article summarizes five aspects of the recent developments of DNA-strand displacement in DNA computing:(1) cascading circuits;(2) catalyzed reaction;(3) logic computation;(4) DNA computing on surfaces; and(5) logic computing based on nanoparticles guided by strand displacement. The applications and mechanisms of strand displacement in DNA computing are discussed and possible future developments are presented.

Toehold-mediated strand displacement reaction-propelled cascade DNAzyme amplifier for microRNA let-7a detection

DNAzyme amplifiers have been extensively explored as a useful sensing platform,but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity.Herein,a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction(TSDR)and Zn^(2+)-assisted DNAzyme cycle with lower cost and simpler procedures.Compared with single DNAzyme amplifier,the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle.Base on this,let-7a could be sensitively detected in the range of 5-50 nmol/L with a detection limit of 64 pmol/L.Furthermore,the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands,which has been successfully applied to the determination of let-7a in blood serum,showing high promise in early cancer diagnosis.

Entropy-driven strand displacement reaction for ultrasensitive detection of circulating tumor DNA based on upconversion and Fe_(3)O_(4) nanocrystals

Early detection of cancer biomarkers applied in real-time disease diagnosis and therapies can increase the survival rate of patients.Circulating tumor DNA(ct DNA)as a typical cancer biomarker plays a great role in the process of tumor disease monitoring,especially in early diagnosis.Unfortunately,most ct DNA detection systems have not been widely used due to their low sensitivity,poor specificity,and high cost.Herein,we developed an alternative ct DNA detection system to present the levels of ct DNA by recording the fluorescence signals of the system containing upconversion nanoparticles(UCNPs),Fe_(3)O_(4),and entropy-driven strand displacement reaction.The method has a practical sensitivity with a wide linear range from 100 amol L^(-1)to 1 nmol L^(-1)and a low detection limit of 1.6 amol L^(-1).Furthermore,the system demonstrates a practical application in mouse blood serum samples and meets the requirements for rapid,sensitive,specific,and economical diagnosis of cancers.Thus,this ct DNA detection system may have great potential for ct DNAdetection and clinical diagnosis.

Cascade Toehold-Mediated Strand Displacement Reaction for Ultrasensitive Detection of Exosomal MicroRNA

MicroRNA(miRNA)in exosomes is a powerful molecular signature for early diagnosis of cancers with the merits of high specificity and high stability.Herein,we report an ultrasensitive electrochemical assay to measure miRNA using a cascade toeholdmediated strand displacement reaction(SDR).In SDR,the trapped exosomal miRNA releases a large amount of single-stranded DNA in the solution.The product then triggers the downstream SDR at the electrode surface.

A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases

Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase(Pol B), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7 d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of Pol B and Pol D from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPf A1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both Pol B and Pol D were efficient in strand displacement. HPf A1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPf A1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

A primer-initiated strand displacement amplification strategy for sensitive detection of 5-Hydroxymethylcytosine in genomic DNA

5-Hydroxymethylcytosine(5 hmC),an intermediate product of DNA demethylation,is important for the regulation of gene expression during development and even tumorigenesis.The challenges associated with determination of 5 hm C level include its extremely low abundance and high structural similarity with other cytosine derivatives,which resulted in sophisticated treatment with large amount of sample input.Herein,we developed a primer-initiated strand displacement amplification(PISDA)strategy to quantify the global 5 hm C in genomic DNA from mammalian tissues with high sensitivity/selectivity,low input and simple operation.This sensitive fluorescence method is based on 5 hmC-specific glucosylation,primer ligation and DNA amplification.After the primer was labeled on 5 hm C site,DNA polymerase and nicking enzyme will repeatedly act on each primer,causing a significant increase of fluorescence signal to magnify the minor difference of 5 hm C content from other cytosine derivatives.This method enables highly sensitive analysis of 5 hm C with a detection limit of 0.003%in DNA(13.6 fmol,S/N=3)from sample input of only 150 ng,which takes less than 15 min for determination.Further determination of 5 hmC in different tissues not only confirms the widespread presence of 5 hmC but also indicates its significant variation in different tissues and ages.Importantly,this PISDA strategy exhibits distinct advantages of bisulfite-free treatment,mild conditions and simple operation without the involvement of either expensive equipment or large amount of DNA sample.This method can be easily performed in almost all research and medical laboratories,and would provide a promising prospect to detect global 5 hmC in mammalian tissues.

Sequence-specific recognition of HIV-1 DNA based upon nicking-assisted strand displacement amplification and triplex DNA

Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus （HIV） infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification （SDA） and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeflciency syndrome （AIDS） in the future.

Recent advances in molecular machines based on toehold-mediated strand displacement reaction

Background： The DNA strand displacement reaction, which uses flexible and programmable DNA molecules as reaction components, is the basis of dynamic DNA nanotechnology, and has been widely used in the design of complex autonomous behaviors. Results： In this review, we first briefly introduce the concept of toehold-mediated strand displacement reaction and its kinetics regulation in pure solution. Thereafter, we review the recent progresses in DNA complex circuit, the assembly of AuNPs driven by DNA molecular machines, and the detection of single nucleotide polymorphism （SNP） using DNA toehold exchange probes in pure solution and in interface state. Lastly, the applications of toehold-mediated strand displacement in the genetic regulation and silencing through combining gene circuit with RNA interference systems are reviewed. Conclusions： The toehold-mediated strand displacement reaction makes DNA an excellent material for the fabrication of molecular machines and complex circuit, and may potentially be used in the disease diagnosis and the regulation of gene silencing in the near future.

Lineal DNA logic gate for microRNA diagnostics with strand displacement and fluorescence resonance energy transfer

Designing molecular logic gates to operate programmably for molecular diagnostics in molecular computing still remains challenging.Here,we designed a novel linear DNA logic gates for microRNA analysis based on strand displacement and fluorescence resonance energy transfer（FRET）.Two labeled strands closed each other produce to FRET through hybridization with a complementary strand to form a basic work unit of logic gate.Two indicators of heart failure（microRNA-195 and microRNA-21） were selected as the logic inputs and the fluorescence mode was used as the logic output.We have demonstrated that the molecular logic gate mechanism worked well with the construction of YES and AND gates.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

New insight into the mechanism of DNA polymerase I revealed by single-molecule FRET studies of Klenow fragment

We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find that there exist in the FRET traces numerous ascending jumps,which are induced by the backsliding of Klenow fragment on DNA chains.Our measurements show that the probability of backsliding is closely related to the GC-richness and d NTP concentration:increasing the GC-richness leads to an increase in the backsliding probability,and increasing the d NTP concentration however leads to a decrease in the backsliding probability.These results provide a new insight into the mechanism of DNA polymerase I.

Simultaneous photoelectrochemical detection of dual microRNAs by capturing CdS quantum dots and methylene blue based on target-initiated strand displaced amplification

Herein,we propose a novel photoelectrochemical(PEC) biosensor for dual microRNAs(miRNAs) highly sensitive and simultaneous biosensing based on strand displaced amplification(SDA) reaction.The recognition of HmiR-21 and Hlet-7 a by microRNA-21 and let-7 a leads to their change in hairpin structures,subsequently initiating the immobilization of abundant CdS quantum dots(CdS QD s) and methylene blue(MB) based on SDA reaction.The immobilized CdS QDs and MB produce both high PEC currents under430 nm light and 627 nm light illumination,respectively,and the generated PEC currents are closely relied on target miRNAs amounts.Thus,highly sensitive and simultaneous detection of microRNA-21 and let-7 a was readily achieved with detection limit at 6.6 fmol/L and 15.4 fmol/L based on 3σ,respectively.Further,this PEC biosensor was applied in simultaneous analysis of miRNA-21 and let-7 a in breast cancer patient’s serum with acceptable results.We expect this biosensor will find more useful application in diagnosis of miRNA-related diseases.

Allosteric DNAzyme-based encoder for molecular information transfer

Dynamic DNA nanotechnology plays a significant role in nanomedicine and information science due to its high programmability based on Watson-Crick base pairing and nanoscale dimensions.Intelligent DNA machines and networks have been widely used in various fields,including molecular imaging,biosensors,drug delivery,information processing,and logic operations.Encoders serve as crucial components for information compilation and transfer,allowing the conversion of information from diverse application scenarios into a format recognized and applied by DNA circuits.However,there are only a few encoder designs with DNA outputs.Moreover,the molecular priority encoder is hardly designed.In this study,we introduce allosteric DNAzyme-based encoders for information transfer.The design of the allosteric domain and the recognition arm allows the input and output to be independent of each other and freely programmable.The pre-packaged mode design achieves uniformity of baseline dynamics and dynamics controllability.We also integrated non-nucleic acid molecules into the encoder through the aptamer design of the allosteric domain.Furthermore,we developed the 2^(n)-n encoder and the EndoⅣ-assisted priority encoder inspired by immunoglobulin's molecular structure and effector patterns.To our knowledge,the proposed encoder is the first enzyme-free DNA encoder with DNA output,and the priority encoder is the first molecular priority encoder in the DNA reaction network.Our encoders avoid complex operations on a single molecule,and their simple structure facilitates their application in complex DNA circuits and biological scenarios.

Ultrasensitive detection of micro RNA-21 in human serum based on the confinement effect enhanced chemical etching of gold nanorods

Natural enzymes,such as horseradish peroxidase(HRP),are a class of important biocatalysts with the high specificity,but their catalytic efficiency is usually unsatisfactory.Thus,the higher catalytic efficiency induced by the confinement effect is promising in optical sensing systems.In this work,a dark-field light scattering sensing platform was fabricated by the confinement effect of HRP from hybridization chain reaction(HCR)and then released to solution by the toehold-mediated strand displacement reaction(TSDR).Then,HRP catalyzed the 3,3,5,5-tetramethylbenzidine(TMB)to TMB^(2+)with the assistance of hydrogen peroxide,which etched the gold nanorods(Au NRs)with the weakened light scattering.The single-particle assay was established based on the decreased light scattering intensity of AuNRs under dark-field microscope.The proposed assay revealed excellent analytical performance within a linear range from 25 pmol/L to 600 pmol/L,and a low limit of detection of 3.12 pmol/L.Additionally,it also manifested satisfactory recovery of mi RNA-21 in human serum samples.The high sensitivity,excellent specificity,and universal applicability make this sensing platform promising for disease diagnosis.