Trigger Strand Research Articles

A Biosensor Based on Interchain Reactions for the Detection of Acetamiprid and the Construction of Basic Logic Gates OR and AND

An enzyme-free and label-free fluorescent DNA aptasensor was constructed with computer assistance based on thermodynamic deviation driving interchain reactions. In this work, in the presence of target acetamiprid, the released trigger strand C-apt could open hairpin Hp1, which in turn triggered the strand displacement reaction and catalyzed the self-assembly of hairpins Hp1 and Hp2, so that the guanine base rich stem in Hp2 was opened. In the presence of K+ and NMM, the G-rich moiety could form a G-quadruplex and emit strong fluorescence at a specific excitation wavelength. The proposed strategy enables sensitive detection of acetamiprid at concentrations as low as 54.3 pM. Most importantly, computer-assisted analysis of the thermodynamic properties of nucleic acid strands and simulation of the reaction process and conditions of the proposed model before conducting biological experiments theoretically proves this strategy feasible and may simplify subsequent biological experiments. In addition, basic molecular logic gates, including OR and AND, were constructed based on this detection principle, and simulation tests and biological experiments were performed. The final results show that this strategy can not only have some applications in the field of food safety and environmental monitoring, but also provide a certain way for the development of molecular logic computing.

A novel fluorescent aptasensor for ultrasensitive detection of Helicobacter pylori in stool samples based on catalytic hairpin assembly cascade hybridization chain reaction

In this work, a fluorescent aptasensor based on enzyme-free catalytic hairpin assembly cascade hybridization chain reaction (CHA-HCR) was established for rapid and sensitive detection of Helicobacter pylori (H. pylori) in stool samples. Four single-stranded DNA hairpins (HP1, HP2, HP3 and HP4), an aptamer and one trigger strand (T strand) were designed, and the stem of HP3 hairpin was modified with a fluorophore (Cy3) and a quencher (BHQ2), respectively. When H. pylori specifically bonded to the aptamer, the T strand was released from aptamer strand. Consequently, the free T strand triggered the CHA-HCR system, and HP2-HP1-[HP3-HP4]n long double strand complex was produced, resulting in the significant fluorescent enhancement. The proposed aptasensor can detect H. pylori in a wide concentration range (20 cfu/mL~ 1000 cfu/mL) with the detection limit as low as 5 cfu/mL. What’s more, the fluorescent aptasensor was implemented to detect H. pylori in 102 stool samples and the results were highly consistent with those of 13C urea breath test (13C-UBT), which demonstrated that the fluorescent aptasensor possessed enormous potential to rapid screening of H. pylori among the whole population and showed novel perspectives in the analysis of other biomarkers, clinical diagnostics, and biomedical applications.

Binding induced isothermal amplification reaction to activate CRISPR/Cas12a for amplified electrochemiluminescence detection of rabies viral RNA via DNA nanotweezer structure switching

Rabies is caused by the infection of Rabies virus, it leads to fatal encephalitis, developing a highly sensitive and specific detection method for Rabies virus remains a challenge. Herein, we report an electrochemiluminescence (ECL) biosensor for Rabies viral RNA based on dual-signal amplification and DNA nanotweezers (DTs). Dual-signal amplification process includes target binding induced isothermal amplification and CRISPR-based amplification. In the presence of target RNA, two assisted probes simultaneously hybridized with it to trigger isothermal amplification with the help of polymerase and nicking enzyme. This process generated a large amount of single-stranded DNA (ssDNA) as products. The products hybridized with CRISPR RNA to activate the trans-cleavage activity of Cas12a to indiscriminately cleave predesigned single-stranded trigger (ST) strands. After mixing the cleavage products with DTs and hemin molecules, DTs cannot be closed by cleaved ST strands to capture hemin to the electrode to quench the ECL signal. Therefore, the higher concentration of the target, the stronger intensity of the ECL signal. The detection limit is as low as 2.8 pM and the detection range is from 5 pM to 5 nM with excellent specificity and stability. The proposed method provides a promising strategy for Rabies detection, and can be easily adapted to other analytes via reasonable design as a valuable and versatile tool in bioanalysis.

Construction of a Homogeneous Enzyme-Free Autocatalytic Nucleic Acid Machinery for High-Performance Intracellular Imaging of MicroRNA

Construction of a Homogeneous Enzyme-Free Autocatalytic Nucleic Acid Machinery for High-Performance Intracellular Imaging of MicroRNA

Enzyme-Free Autocatalysis-Driven Feedback DNA Circuits for Amplified Aptasensing of Living Cells.

Aptasensors with high specificity have emerged as powerful tools for understanding various biological processes, thus providing tremendous opportunities for clinical diagnosis and prognosis. However, their applications in intracellular molecular imaging are largely impeded due to the low anti-interference capacity in biological environments and the moderate sensitivity to targets. Herein, a robust enzyme-free autocatalysis-driven feedback DNA circuit is devised for amplified aptasensing, for example, adenosine triphosphate (ATP) and thrombin, with a significantly improved sensitivity in living cells. This initiator-replicated hybridization chain reaction (ID-HCR) circuit was acquired by integrating the HCR circuit with the DNAzyme biocatalysis. Also, the autocatalysis-driven aptasensor consists of a recognition element and an amplification element. The recognition unit can specifically identify ATP or thrombin via a versatile conformational transformation, resulting in the exposure of the initiator to the autocatalysis-driven circuit. The ID-HCR element integrates the charming self-assembly characteristics of the HCR and the remarkable catalytic cleavage capacity of DNAzyme for realizing the continuously self-sustained regeneration or replication of trigger strands and for achieving an exponential signal gain. The autocatalysis-driven aptasensor has been validated for quantitative analysis of ATP and thrombin in vitro and for monitoring the corresponding aptamer substrates with various expressions in live cells. More importantly, the autocatalysis-driven aptasensor, as a versatile amplification strategy, holds enormous potential for analysis of other less abundant biomarkers by changing only the recognition element of the system.

Controlled Release in Hydrogels Using DNA Nanotechnology.

Gelatin is a biopolymer widely used to synthesize hydrogels for biomedical applications, such as tissue engineering and bioinks for 3D bioprinting. However, as with other biopolymer-based hydrogels, gelatin-hydrogels do not allow precise temporal control of the biomolecule distribution to mimic biological signals involved in biological mechanisms. Leveraging DNA nanotechnology tools to develop a responsive controlled release system via strand displacement has demonstrated the ability to encode logic process, which would enable a more sophisticated design for controlled release. However, this unique and dynamic system has not yet been incorporated within any hydrogels to create a complete release circuit mechanism that closely resembles the sequential distribution of biomolecules observed in the native environment. Here, we designed and synthesized versatile multi-arm DNA motifs that can be easily conjugated within a gelatin hydrogel via click chemistry to incorporate a strand displacement circuit. After validating the incorporation and showing the increased stability of DNA motifs against degradation once embedded in the hydrogel, we demonstrated the ability of our system to release multiple model cargos with temporal specificity by the addition of the trigger strands specific to each cargo. Additionally, we were able to modulate the rate and quantity of cargo release by tuning the sequence of the trigger strands.

Disassembly of DNA origami dimers controlled by programmable polymerase primers.

Dynamic regulation of DNA origami nanostructures is important for the fabrication of intelligent DNA nanodevices. Toehold-mediated strand displacement is a common regulation strategy, which utilizes trigger strands to assemble and disassemble nanostructures. Such trigger strands are required to be completely complementary to the corresponding substrate strands, which strictly demands orthogonality and accuracy of the sequence design. Herein, we present a disassembly strategy of DNA origami dimers based on polymerase-triggered strand displacement, where the polymerase primers, as the trigger strands, were only partially complementary to the toehold region of the substrate strands. To demonstrate the programmability of trigger strands, we utilized primers with different sequence combination patterns to disassemble DNA origami dimers. The statistical summary of AFM images and fluorescence curves proved the feasibility of the new strategy. The utilization of polymerase-triggered strand displacement on the disassembly of DNA origami structures enriches the toolbox for the dynamic regulation of DNA nanostructures.

Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription–free exponential amplification reaction, RTF-EXPAR

A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.

Regulation of reversible conformational change, size switching, and immunomodulation of RNA nanocubes.

In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility in structure, diversity in function, and adaptability in a configuration similar to proteins. In the past, the work on the investigation of conformational change mainly focused on protein. The induced-fit and conformational capture in RNA have also been explored, such as in the study of riboswitches. Herein, we report the engineering of three-dimensional RNA nanocubes and demonstrated the operation and regulation for its configuration. We demonstrate the operation of reconfigurable RNA nanocubes whose shapes change precisely and reversibly in response to a specific trigger strand. The shape, size, and conformation can be regulated precisely and reversibly in response to the specific triggering signals. The shape and conformational conversion were observed by cryo-EM and gel electrophoresis, respectively. Harnessing the size, shape, conformation, and self-assembly capabilities of the RNA nanocube can provide a new potential use of this technology as nanocarriers for the treatment of various diseases.

A cascade strand displacement amplification strategy for highly sensitive and label-free detection of DNA methyltransferase activity

DNA methyltransferase (MTase) is regarded as a promising biomarker and therapeutic target, which plays an important regulatory role in many biological processes. Here, a cascade strand displacement amplification strategy was explored for highly sensitive and label-free fluorescent detection of DNA MTase activity. Firstly, the hairpin recognition probe (RP) with a long stem was designed. DNA MTase catalyzed the RP methylation, and then DpnI endonuclease cleaved the methylated RP, releasing a trigger strand. Subsequently, it as a primer hybridized with hairpin probe 1 and triggered strand displacement amplification reaction (SDAR), producing numerous replaced strands. And the replaced strands hybridized with hairpin probe 2 and triggered next SDAR, producing a larger number of G-rich sequences. After that, N-methylmesoporphyrin IX was selectively intercalated into the G-quadruplex structures which were folded from the G-rich sequences, obtaining a significantly enhanced fluorescent signal. However, RP was stable, and could not produce primer for the subsequent cascade SDAR in the absence of DNA MTase. The effective amplification efficiency of cascade SDAR improved the detection sensitivity (detection limit: 0.0073 U/mL). Moreover, this strategy well evaluated the inhibitions of 5-fluorouracil, benzylpenicillin and gentamycin on DNA MTase activity. The results indicated this method held great potential in new method exploration and early disease diagnosis.

Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions.

A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.

Modular Reconfigurable DNA Origami: From Two-Dimensional to Three-Dimensional Structures.

DNA origami enables the manipulation of objects at nanoscale, and demonstrates unprecedented versatility for fabricating both static and dynamic nanostructures. In this work, we introduce a new strategy for transferring modular reconfigurable DNA nanostructures from two-dimensional to three-dimensional. A 2D DNA sheet could be modularized into connected parts (e.g., two, three, and four parts in this work), which can be independently transformed between two conformations with a few DNA "trigger" strands. More interestingly, the transformation of the connected 2D modules can lead to the controlled, resettable structural conversion of a 2D sheet to a 3D architecture, due to the constraints induced by the connections between the 2D modules. This new approach can provide an efficient mean for constructing programmable, higher-order, and complex DNA objects, as well as sophisticated dynamic substrates for various applications.

Multiplex monitoring of Alzheimer associated miRNAs based on the modular logic circuit operation and doping of catalytic hairpin assembly

In recent years, DNA logic gates have been extensively applied in developing multiplex processing platforms to provide an accurate decision on the diagnosis of multi-factor diseases. In this work, we presented a new cascaded logical operator by combining different modules for computational monitoring of four miRNAs related to Alzheimer disease (has-miR-143-3p, has-miR-18b-5p, has-miR-424-5p, and has-miR-93-5p). Herein, three sequential logic gates were programed that upon entering the miRNA inputs, delivered the trigger strand of CHA (catalytic hairpin assembly) reaction through a cyclic amplification. Afterward, the product of the CHA reaction, three-way junction, could induce the gold nanoparticles aggregation. This phenomenon led to generate a blue color of the solution that enabled visualizing and quantitative measurement of the assay. The output signals were recorded through reading the absorbance intensity transition, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Taken together, the proposed assay by taking advantage of excellent generality, naked eye observation, the 4-plex detection, simplicity, enzyme-free nature, and two-steps process without any immobilization and washing has addressed the limitation of the previous systems. Moreover, the amplified monitoring of low-abundant of target miRNAs was accomplished with a limit of detection as low as 5 pM.

CRISPR-Cas9 Triggered Two-Step Isothermal Amplification Method for E. coli O157:H7 Detection Based on a Metal-Organic Framework Platform.

Escherichia coli O157:H7 has been reported as an important pathogenic bacteria causing serious infection and economic loss. However, detection of Escherichia coli O157:H7 needs improvement, given its current complexity and sensitivity. Herein, we attempt to build a fluorescence sensing method to detect Escherichia coli O157:H7 with easy operation and high efficiency. The target virulence gene sequences are recognized and cleaved by the CRISPR-Cas9 system, and trigger strand displacement amplification and rolling circle amplification. After amplification reactions, massive products can hybridize with the probes, the fluorescence of which are quenched based on a metal-organic framework platform, leading to the fluorescence recovery at typical excitation/emission wavelengths of 480/518 nm. This method exhibits high sensitivity with the detection limit at 4.0 × 101 CFU mL-1 and a wide range from 1.3 × 102 CFU mL-1 to 6.5 × 104 CFU mL-1. Meanwhile, this assay also shows significant specificity and applies to practical samples with high accuracy. Therefore, our method would have great potential application in bacterial detection, food safety monitoring, or clinical diagnostics.

Three-way junction-induced isothermal amplification for nucleic acid detection

We, herein, describe a three-way junction (3WJ)-induced isothermal amplification (ThIsAmp) reaction for target nucleic acid detection. In this strategy, target nucleic acid induces the formation of 3WJ structure by associating a specially designed ThIsAmp template and ThIsAmp primer. Upon the formation of 3WJ structure, ThIsAmp primer is subjected to continuously repeated extension and nicking reaction by the combined activities of DNA polymerase and nicking endonuclease, consequently producing a large number of trigger strands. The trigger strands then initiate two separate but interconnected pathways by binding to either 3’ overhang of ThIsAmp template within the 3WJ structure or free ThIsAmp template. As a consequence, a large number of final double-stranded DNA products are produced under an isothermal condition, which can be monitored in real-time by detecting the fluorescence intensity resulting from SYBR Green I staining. Based on this principle, we successfully detected target DNA down to 78.1 aM with excellent specificity. The sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling target nucleic acid detection.

Novel 3D Printed Device for Dual-Signaling Ratiometric Photoelectrochemical Readout of Biomarker Using λ-Exonuclease-Assisted Recycling Amplification.

A ratiometric photoelectrochemical (PEC) sensing strategy was proposed for monitoring of carcinoembryonic antigen (CEA) based on a homemade 3D printing device with dual-working photoelectrodes (PE1 and PE2), coupling λ-exonuclease (λ-Exo)-assisted recycling amplification with CdS quantum dots. Gold nanoparticles-functionalized ZnO nanorods were utilized as PEC substrate for generating initial photocurrent and immobilizing DNA probe. Upon incubation of target with DNA trigger/CEA aptamer-modified magnetic bead (tri/apt-MB), DNA trigger dissociated from magnetic bead and then hybridized with capture probe (cp) on PE1 or opened hairpin probe (hp) on PE2 to form double-stranded DNA (dsDNA). The exonuclease could recognize and cleave two newly generated dsDNA, leading to the release of trigger. The free trigger strand continued to hybridize with the remaining cp/hp, which were cleaved by λ-Exo, and then trigger was released again and restarted next recycle with the λ-Exo. After digestion of λ-Exo, the number of capture probes on PE1 was reduced, and many short DNA fragments were produced on PE2, thereby resulting in the decreasing CdS QDs on PE1 and the increasing CdS QDs on PE2. As a result, it was observed that the ratio value of photocurrents (PE1/PE2) significantly decreased with the increasing CEA. Under optimum conditions, the sensing method showed a good linear relationship toward CEA within the dynamic range of 0.02-10 ng mL-1 and a detection limit of 6.0 pg mL-1. Moreover, the ratiometric PEC sensor exhibited good reproducibility, satisfying stability, and remarkable anti-interference performance, which suggests its promising application prospect to detect target CEA.

Exonuclease III-assisted strand displacement reaction-driven cyclic generation of G-quadruplex strategy for homogeneous fluorescent detection of melamine

Development of flexible, sensitive, selective and simple melamine detection is in high demand and of great significance. Here, we proposed a homogeneous turn-off mode fluorescent strategy to detect melamine by coupling the process of exonuclease III-assisted (Exo III) amplification with the strand displacement reaction (SDR)-driven assembly of DNA G-quadruplex structures. Melamine (M) could bind to the thymine (T) base through hydrogen bonding to form a T-M-T structure, which inhibits the subsequent nucleic acid amplification reaction and formation of the G-quadruplex structure. DNA strand (G1) served as both a recognition probe and a signal probe, thus greatly simplifying the experimental design and operation. Furthermore, to improve the detection sensitivity, an ingeniously designed segment of double-stranded DNA (dsDNA, P1-G2) that contained the G-quadruplex sequence (G2) and a partially hybridized signal trigger strand (P1) was added to the system. Following the SDR and Exo III-assisted amplification, G1-DNA and G2-DNA were released and the cyclic production of G-quadruplex structures was initiated. The generated DNA G-quadruplex bound to the NMM (a fluorescent dye, N-methyl mesoporphyrin IX), providing enhanced fluorescence signals. This allowed for the highly sensitive detection of melamine at concentrations as low as 25 fM. The proposed fluorescent detection method of melamine concentration displayed a good linear relationship ranging from 100 fM to 100 pM, which showed improved performance over that of the non-enzyme-assisted sensing system (limit of detection is 15 pM). Additionally, this method exhibited a high selectivity towards the target molecule, melamine, in comparison to other substances. Furthermore, the method was used for the assay of melamine in real milk samples and satisfactory experiment results were obtained. The novel strategy developed in this research may be considered as a potential route for highly sensitive, simple, selective and accurate assay of small molecules.

Design and operation of reconfigurable two-dimensional DNA molecular arrays.

Information relay and cascaded transformation are essential in biology and engineering. Imitation of such complex behaviors via synthetic molecular self-assembly at the nanoscale remains challenging. Here we describe the use of structural DNA nanotechnology to realize prescribed, multistep, long-range information relay and cascaded transformation in rationally designed molecular arrays. The engineered arrays provide a controlled platform for studying complex dynamic behaviors of molecular arrays and have a range of potential applications, such as with reconfigurable metamaterials. A reconfigurable array consists of a prescribed number of interconnected dynamic DNA antijunctions. Each antijunction unit consists of four DNA domains of equal length with four dynamic nicking points, which are capable of switching between two stable conformations through an intermediate open conformation. By interconnecting the small DNA antijunctions, one can build custom two-dimensional (2D) molecular 'domino' arrays with arbitrary shapes. More important, the DNA molecular arrays are capable of undergoing programmed, multistep, long-range transformation driven by information relay between neighboring antijunction units. The information relay is initiated by the trigger strands under high temperature or formamide concentration. The array's dynamic behavior can be regulated by external factors such as its shape and size, points of transformation initiation, and/or any engineered information propagation pathways. This protocol provides detailed strategies for designing DNA molecular arrays, as well as procedures for sample production, purification, reconfiguration, and imaging by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The procedure can be completed in 4-7 d.

Target-programmed and autonomous proximity binding aptasensor for amplified electronic detection of thrombin

The development of sensitive and simple approaches capable of monitoring trace amounts of protein biomarkers is appealing for disease diagnosis and treatment. Towards this end, we have developed an electrochemical sensing platform for sensitive and simple detection of protein biomarkers by using thrombin as the model target molecules via a target-programmed proximity binding amplification approach. The binding of thrombin to the aptamer sequences in the partial dsDNA duplex probes induces the release of the ssDNA trigger strands, which catalyze subsequent assembly formation of many methylene blue (MB)-tagged proximate DNA motifs with the presence of the DNA fuel strands through cascaded toehold-mediated strand displacement reactions. Due to the proximity-binding effect, these MB-tagged proximate DNA motifs anneal with the capture probes on the sensor surface with significantly enhanced stability against the corresponding single component counterpart, thereby pulling the MB tags close to the sensor surface and generating substantially amplified signal responses for sensitive determination of thrombin down to 23.6 pM. In addition, such aptasensor can specifically discriminate thrombin from other interference proteins, and can also be utilized to monitor thrombin in diluted serum samples, demonstrating its great potential for sensitive determination of proteins for early disease diagnosis.

Rapid Photoactuation of a DNA Nanostructure using an Internal Photocaged Trigger Strand.

A reconfigurable DNA nano-tweezer is reported that can be switched between a closed and open state with a brief pulse of UV light. In its initial state, the tweezer is held shut using a hairpin with a single-stranded poly-A loop. Also incorporated in the structure is a poly-T trigger strand bearing seven photocaged residues. Upon illumination with 365 nm light, the cages are removed and the trigger strand hybridizes to the loop, opening the tweezer and increasing the distance between its arms from 4 to 18 nm. This intramolecular process is roughly 60 times faster than adding an external trigger strand, and provides a mechanism for the rapid interconversion of DNA nanostructures with light.