Catalytic Hairpin Assembly Research Articles

Lighting up Lipidic Nanoflares with Self-Powered and Multivalent 3D DNA Rolling Motors for High-Efficiency MicroRNA Sensing in Serum and Living Cells.

Intelligent DNA nanomachines are powerful and versatile molecular tools for bioimaging and biodiagnostic applications; however, they are generally constrained by complicated synthetic processes and poor reaction efficiencies. In this study, we developed a simple and efficient molecular machine by coupling a self-powered rolling motor with a lipidic nanoflare (termed RMNF), enabling high-contrast, robust, and rapid probing of cancer-associated microRNA (miRNA) in serum and living cells. The lipidic nanoflare is a cholesterol-based lipidic micelle decorated with hairpin-shaped tracks that can be facilely synthesized by stirring in buffered solution, whereas the 3D rolling motor (3D RM) is a rigidified tetrahedral DNA scaffold equipped with four single-stranded "legs" each silenced by a locking strand. Once exposed to the target miRNA, the 3D RM can be activated, followed by self-powered precession based on catalyzed hairpin assembly (CHA) and lighting up of the lipidic nanoflare. Notably, the multivalent 3D RM that moves using four DNA legs, which allows the motor to continuously and acceleratedly interreact with DNA tracks rather than dissociate from the surface of the nanoflare, yielded a limit of detection (LOD) of 500 fM at 37 °C within 1.5 h. Through the nick-hidden and rigidified structure design, RMNF exhibits high biostability and a low false-positive signal under complex physiological settings. The final application of RMNF for miRNA detection in clinical samples and living cells demonstrates its considerable potential for biomedical imaging and clinical diagnosis.

A target induced self-powered catalyzed hairpin assembly coupled with DNA motor amplification for miRNA detection in colorectal cancer

A target induced self-powered catalyzed hairpin assembly coupled with DNA motor amplification strategy was presented for miRNA-92a detection. The miRNA is used to open the hairpin and initiate the catalyzed hairpin assembly (CHA). The product of CHA can catalyze the CHA reaction and cause the recovery of fluorescent intensity. The limit of detection (LOD) could reach to 0.04 fM with good linear range from 0.2 fM to 500 fM. Importantly, the time of two level amplification reaction was greatly shortened to 130 min by the high efficiency of self-powered CHA strategy. The amplification efficiency of self-powered CHA strategy was investigated. It also showed great selectivity and specificity for other miRNA sequences. In addition, it exhibited satisfactory results for practical application.

Rationally Designed Dual Base Pair Mismatch Enables Toehold-Mediated Strand Displacement to Efficiently Recognize Single-Nucleotide Polymorphism without Enzymes.

The efficiency of the enzyme-free toehold-mediated strand displacement (TMSD) technique is often insufficient to detect single-nucleotide polymorphism (SNP) that possesses only single base pair mismatch discrimination. Here, we report a novel dual base pair mismatch strategy enabling TMSD biosensing for SNP detection under enzyme-free conditions when coupled with catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET). The strategy is based on a competitive strand displacement reaction mechanism, affected by the thermodynamic stability originating from rationally designed dual base pair mismatch, for the specific recognition of mutant-type DNA. In particular, enzyme-free nucleic acid circuits, such as CHA, emerge as a powerful method for signal amplification. Eventually, the signal transduction of this proposed biosensor was determined by FRET between streptavidin-coated 605 nm emission quantum dots (605QDs, donor) and Cy5/biotin hybridization (acceptor, from CHA) when incubated with each other. The proposed biosensor displayed high sensitivity to the mutant target (MT) with a detection concentration down to 4.3 fM and led to high discrimination factors for all types of mismatches in multiple sequence contexts. As such, the application of this proposed biosensor to investigate mechanisms of the competitive strand displacement reaction further illustrates the versatility of our dual base pair mismatch strategy, which can be utilized for the creation of a new class of biosensors.

Chemiluminescent plate assay of microRNA-155 coupled with catalytic hairpin assembly with oligonucleotide release (CHAOR)

A heterogeneous sensitive microRNA-155 assay based on a new isothermal amplification method, called catalytic hairpin assembly with oligonucleotide release (CHAOR), was developed. The principle of CHAOR was studied by non-denaturing electrophoresis. To detect the amplification product, a polyperoxidase-streptavidin conjugate (molar ratio 1:80) and an enhanced chemiluminescence reaction were used, which made it possible to increase assay sensitivity. The detection limit of microRNA-155 assay was 0.4 pM. The coefficient of variation of the chemiluminescent signal, formed upon the heterogeneous determination of miRNA-155, was less than 12 % within the working range. The efficiency of CHAOR as an amplification method was similar to that of traditional CHA, as miRNA-155 assays based on CHAOR and CHA had similar analytical parameters. In addition, the proposed assay was highly specific. Contrary to traditional CHA, CHAOR, one of whose products is a single-stranded oligonucleotide, can be used in analytical methods based on cascade amplification.

Self-assembled programmable DNA nanoflower for in situ synthesis of gold nanoclusters and integration with Mn-MOF to sensitively detect AFB1

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that is susceptible to contamination of agricultural products and can cause chronic effects on humans. Thus, the establishment of highly sensitive and efficient AFB1 detection method is crucial to ensure food safety and human health. Here, we developed programmable DNA nanoflowers (DNF) with dual functionality for in situ synthesis of gold nanoclusters (AuNCs) and DNA recognition. The AuNCs synthesized using DNF as template (DNF@AuNCs) possessed good fluorescence properties and stability, and were applied as stable signal probes for constructing sensitive aptasensor. DNF completed the transition from bud to blooming flower, shifting its morphology from 3D to 2D structure during the in-situ synthesis of AuNCs, leading to the exposure of more template sequences. The designed hairpins DNA were subsequently used for local catalytic hairpin assembly (LCHA) on the surface of Mn-MOF. A substantial number of amplified products H2-H3 was obtained by conformational conversion, which was complementary to the sequence of DNF. Upon hybridization of H2-H3 with DNF@AuNCs, the fluorescence of AuNCs is quenched by adjacent guanine-rich (G-rich) base at the 3′ end of H3. The switch of LCHA is controlled by the conformational change of H1 which contains the AFB1 aptamer sequence, thus enabling precise detection of AFB1. The DNF@AuNCs and Mn-MOF-based fluorescence aptasensor achieved a detection limit of 7 pg/mL for AFB1, with a detection range of 0.01–200 ng/mL. The aptasensor also exhibits high selectivity, reproducibility, and stability, making it prospective for food safety monitoring.

Actively targeted and dual-stimuli-responsive branch-shaped system for simultaneous microRNAs imaging in living cells

Designing a multifunctional nucleic acid platform with the ability to identify multiple biomarkers is crucial for accurate molecular diagnostics. Here we reported a dual-target branch-shaped nucleic acid probing (DBNP) system and its targeted delivery for simultaneous analysis of intracellular microRNAs (miRNAs). The system consisted of a cross-shaped DNA scaffold equipped with four hairpin strands and an aptamer strand. The four hairpin strands constituted two catalytic hairpin assembly (CHA) reactions, enabling the system to simultaneously recognize and amplify two kinds of miRNAs. We demonstrated that the cross-shaped DNA scaffold structurally offered a spatial-confinement effect, well-known in increasing the intramolecular collision of the probes and accelerating the CHA reactions, which allows us to resolve multiplex miRNAs profiling with high efficiency by using DBNP system. In addition, because of the inherent character of aptamer capable of specifically binding with cell-membrane receptor, targeted delivery of DBNP system into the unique receptor-positive cells can be realized to detect intracellular miRNAs. The dual miRNA-mediated stimuli-response, operating in parallel with a receptor-specific recognition of target cells, synergistically enhances the accuracy of cell discrimination and cancer diagnosis. This innovative approach holds great promise for advancing the development of cutting-edge signal amplification technologies and biosensors utilized in cancer diagnostics.

3D-DNA walking nanomachine based on catalytic hairpin assembly and copper nanoclusters for sensitive detection of hepatitis C virus

Rapid and accurate detection of the hepatitis C virus (HCV) is essential for early diagnosis and prevention of virus transmission. This study presents a novel approach that combines the three-dimensional (3D)-DNA walking nanomachine with catalytic hairpin assembly (CHA) and copper nanoclusters (CuNCs). By integrating CHA with the 3D DNA walking nanomachine, efficient target amplification on 3D surfaces was achieved, leading to improved reaction speed and detection performance. Terminal deoxynucleotidyl transferase (TdT) was utilized to generate T-rich DNA sequences. These sequences served as templates for the formation of CuNCs, which functioned as the readout signal. The optimized 3D-DNA walking nanomachine exhibited excellent sensitivity in detecting HCV, with a detection limit of 42.4 pM and a linear range of 100 pM to 2 nM. The biosensor demonstrated excellent selectivity and reproducibility, with a recovery rate ranging from 94% to 108% for the detection of real samples. This design holds great potential for sensitive, label-free, and reliable detection of HCV in clinical settings. Furthermore, the versatility of this approach allows for the customization of target sequences, thereby facilitating the detection of various nucleic acid targets. Therefore, this method has the potential to advance personalized medicine, disease management, and genetic analysis in the field of molecular diagnosis.

A novel feedback catalytic hairpin assembly strategy for sensitive mRNA imaging in living cells

Achieving specific and sensitive imaging of mRNA in living cells plays a key role in cancer diagnosis and treatment. Catalytic hairpin assembly (CHA) has been widely used for mRNA imaging. However, it has been significantly constrained by limited in-depth amplifications. Herein, a novel feedback catalytic hairpin assembly (FCHA) with exponential amplification efficiency was proposed for mRNA imaging in living cells. The FCHA system consisted of with two hairpin DNAs (H1, H2) and a double stranded DNA (H3L). After the probes enter the cell by using β-FeOOH nanoparticles (NPs) as delivery vector, the intracellular target Tk1 mRNA acted as catalyst to trigger the FCHA between H1, H2 and H3L, which achieved the exponential growth of CHA product. This FCHA system has demonstrated improved reaction kinetics and sensitivity compared to traditional CHA, which was 106 times more sensitive than conventional CHA. This strategy can be used for real-time imaging and indication of changes in the mRNA expression. This strategy slao can distinguish the expression difference of mRNA in normal and tumor cells, and can evaluate the anti-tumor drug’s efficacy. Benefiting from these outstanding advantages, we believe that this FCHA will become a powerful tool for early cancer diagnosis.

CRISPR-Cas12a powered hybrid nanoparticle for extracellular vesicle aggregation and in-situ microRNA detection

Efficient extracellular vesicle (EV) enrichment and timely internal RNA detection for cancer diagnostics are highly desirable and remain a challenge. Here, we report a rapid EV aggregation induced in-situ microRNA detection technology based on cationic lipid-polymer hybrid nanoparticles encapsulating cascade system of catalytic hairpin assembly and CRISPR-Cas12a (CLHN-CCC), allowing for EV enrichment in three-dimensional space and in-situ detection of internal microRNAs in one step within 30 min. The enrichment efficiency (>90%) of CLHN-CCC is demonstrated in artificial EVs, cell-secreted EVs and serum EVs, which is 5-fold higher than that of traditional ultracentrifugation. The sensitive detection of artificial EVs and internal miR-1290 was achieved with the limit of detection of 10 particles/μL and 0.07 amol, respectively. After lyophilization, CLHN-CCC shows no obvious loss of performance within 6 months, making it much more robust and user friendly. This technique could sensitively (sensitivity = 92.9%) and selectively (selectivity = 85.7%) identify low amount miR-1290 in serum EVs, distinguishing early-stage pancreatic cancer patients from healthy subjects, showing high potential for clinical applications.

Highly sensitive miRNA detection of early-stage laryngeal carcinoma using a solid-state Au nanocone arrays fabricated LoC-SERS analysis system coupled with target-triggered dual cycle amplification strategy

Herein, with a target-triggered dual cycle amplification strategy, a novel lab-on-a-chip-based surface-enhanced Raman scattering (LoC-SERS) analysis system was established to analyze miRNAs quantitatively. Due to a more substantial complementary pairing effect, the presence of targets can replace the single-stranded DNA S1 (S1) from the single-stranded DNA F1 (F1) and single-stranded DNA S2 (S2) can replace and release the targets similarly. Thus, target can cyclically trigger the cycle Ⅰ and release S1 continuously. Cycle Ⅱ is the catalyzed hairpin assembly (CHA) event between the hairpin DNA 1 and hairpin DNA 2, which can connect the Ag-Au nanorods (Ag-AuNRs) to the surface of solid-state Au nanocone arrays (AuNCAs). Owing the dual cycle amplification strategy, localized surface plasmon resonance (LSPR) can be excited and significantly magnify the local electromagnetic field for SERS signal enhancement. Thus, ultrasensitive detection of miR-21 and miR-106b was achieved with the limit of detection as low as aM level. Attributed to the hydrophilic treatment and the design of capillary pump, the detection can be finished without any external pumps. Moreover, the system exhibited good practicability in the analysis of real serum samples obtained from Laryngeal carcinoma (LC) patients and healthy subjects. Therefore, the system is a promising candidate in clinical application and exhibited potential for the prediction, diagnosis, monitoring, and staging of LC.

Balancing sensitivity, speed, and accuracy in miRNA detection with a localized branched catalytic hairpin assembly strategy

Developing accurate, sensitive, and rapid methods for microRNA analysis in living cells is crucial for cancer diagnosis. Recent advances in enzyme-free amplification and spatial-confinement techniques have enhanced both the sensitivity and speed of miRNA detection. However, they also introduce a challenging contradiction: while localized amplification increases the sensitivity of miRNA detection, the consequent elevated risk of probe crosstalk can lead to false-positive signals and compromised accuracy. To strike a balance between the sensitivity, speed, and accuracy, we herein propose a localized branched catalytic hairpin assembly (LBCHA) strategy. This method employs three independent nanoprobes, each equipped with a specific branched catalytic hairpin assembly (bCHA) hairpin. The presence of the target initiates localized bCHA among the LRN probes, generating amplified fluorescent signals. Importantly, by anchoring each bCHA hairpin on separate nanospheres, we enhance detection sensitivity and speed while preserving accuracy by minimizing non-specific interactions. The LBCHA system demonstrated exceptional miRNA sensing performance in vitro, reaching a detection limit of 3.66 pM. Further applications in live cells and clinically derived tissues confirmed its potential for accurate cancer discrimination. This innovative LBCHA strategy holds great potential for clinical applications and real-time cancer diagnostics.

Development of a regenerable dual-trigger tripedal DNA walker electrochemical biosensor for sensitive detection of microRNA-155

Since microRNAs (miRNAs) are valuable biomarkers for disease diagnosis and prognosis, the pursuit of enhanced detection sensitivity through signal amplification strategies has emerged as a prominent focus in low-abundance miRNA detection research. DNA walkers, as dynamic DNA nanodevice, have gained significant attention for their applications as signal amplification strategies. To overcome the limitations of unipedal DNA walkers with a restricted signal amplification efficiency, there is a great need for multi-pedal DNA walkers that offer improved walking and signal amplification capabilities. Here, we employed a combination of catalytic hairpin assembly (CHA) and APE1 enzymatic cleavage reactions to construct a tripedal DNA walker, driving its movement to establish a cascade signal amplification system for the electrochemical detection of miRNA-155. The biosensor utilizes tumor cell-endogenous microRNA-155 and APE1 as dual-trigger for DNA walker formation and walking movement, leading to highly efficient and controllable signal amplification. The biosensor exhibited high sensitivity, with a low detection limit of 10 pM for microRNA-155, and successfully differentiated and selectively detected microRNA-155 from other interfering RNAs. Successful detection in 20 % serum samples indicates its potential clinical application. In addition, we harnessed strand displacement reactions to create a gentle yet efficient electrode regeneration strategy, to addresses the time-consuming challenges during electrode modification processes. We have successfully demonstrated the stability of current signals even after multiple cycles of electrode regeneration. This study showcased the high-efficiency amplification potential of multi-pedal DNA walkers and the effectiveness and versatility of strand displacement in biosensing applications. It opens a promising path for developing regenerable electrochemical biosensors. This regenerable strategy for electrochemical biosensors is both label-free and cost-effective, and holds promise for detecting various disease-related RNA targets beyond its current application.

Movable toehold for leakless self-assembly circuits

Nonenzymatic self-assembly circuit utilizing hairpin substrates has been developed to be a powerful tool for information transduction, amplification and computation. However, the sensitivity, stability and application of this circuit are impeded by the presence of leakage which refers to undesired triggering in the absence of input. Herein, we proposed a movable toehold principle to suppress leakage and accelerate the catalytic reaction through removing partial hairpin toehold responsible for the leakage and transferring it to the catalyst. With movable toehold, catalytic hairpin assembly (called mtCHA) exhibited an excellent signal-to-background ratio of over 100, high robustness and improved specificity. In more complex circuit, including proximity recognition, signal amplification of small molecules (such as ATP), logic network, autocatalysis circuit and two-layer cascade circuit, mtCHA also demonstrated satisfactory performance. Our findings suggest that mtCHA holds great potential for broader applications, and the approach of repurposing harmful fragments into beneficial candidates can provide valuable insights for other chemical systems.

Catalytic hairpin assembly-mediated SERS biosensor for double detection of MiRNAs using gold nanoclusters-doped COF substrate

A novel surface-enhanced Raman scattering (SERS) and catalytic hairpin assembly (CHA) technique dependent dual-signal amplification method for double detection of microRNAs (miRNAs) was created in this work. When target miRNAs were present, CHA reactions could be triggered between the corresponding hairpin DNAs, fixing the SERS tags (two distinct Raman reporters, 4-mercaptobenzonitrile and 4-mercaptopyridine, which were pre-labeled in the space between Au core and silver shell nanoparticles) onto the gold nanoclusters-doped covalent organic frameworks (AuNCs/COF) nanofiber substrate surface, which generated numerous “hot spots” and ultimately produced amplified SERS signals. Besides, with the help of the CHA cycles, much more target miRNAs were captured to improve the detection sensitivity and decrease the detection limit of multiplex detection down to 77 aM for miRNA-21 and 93 aM for miRNA-155, respectively. This SERS biosensor also exhibited a broad detection range (10−16 −10−12 M), good specificity, high reproducibility, as well as excellent recoveries in spiked human serum samples (in the range of 94.5–99.4%). The findings of our experiment suggested that the multiple signal amplification biosensors that were developed have significant potential for multiplex detection of cancer biomarkers (miRNAs and their analogs) in the fields of bioanalysis and clinical biomedicine.

A biosensor using semi-DNA walker and CHA -FRET loop for ultrasensitive detection of single nucleotide polymorphism

Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. However, it is still challenging to make highly specific and sensitive SNPs detection to distinguish target DNA with single-base difference, so that flap endonuclease 1 (FEN 1) manipulated semi-DNA walker reaction and catalytic hairpin assembly (CHA)-FRET loop for ultrasensitive SNP detection has been proposed. Herein, FEN 1 was employed to specifically recognize and cut the 5′ flap of three-base overlapping structure formed by only hybridizing with mutant target (MT). Simultaneously, FEN 1 drove MT hybridization to autonomously walk along downstream probe (DP) and the cut sequence (CS) from semi-DNA walker reaction was collected by magnetic bead-based separation to avoid false-positive signal. Next, the cleaved CS triggered CHA and generated fluorescence resonance energy transfer (FRET) via Cy3 and Cy5. FEN 1 manipulated semi-DNA walker reaction to specifically recognize the target DNA and combined CHA-FRET loop to dually amplify signal. Thus, the SNP biosensor demonstrated superior performance for ultrasensitive detection of mutant KRAS gene with a low detection limit as low as 80 aM and was successfully applied to monitor the expression of mutant KRAS gene in human cancer cell lysates. This strategy provides an ultrasensitive way for the detection of biomolecules and reveals an effective avenue for diseases diagnosis.

Ag+-stabilized DNA triplex coupled with catalytic hairpin assembly and CRISPR/Cas12a amplifications for sensitive metallothionein assay

Metallothionein (MT) is a protein biomarker secreted by liver in response to the treatment for heavy metal toxicity and oncological diseases. On the basis of a new Ag+-stabilized DNA triplex probe (Ag+-SDTP), we establish a fluorescent biosensing system for high sensitivity detection of MT by combining catalytic hairpin assembly (CHA) and the CRISPR/Cas12a signal enhancements. The MT analyte complexes with Ag+ in Ag+-SDTP to disrupt the triplex structure and to release the ssDNA strands, which trigger subsequent CHA formation of many protospacer adjacent motif (PAM)-containing dsDNAs from two hairpins. Cas12a/crRNA further recognizes these PAM sequences to activate its trans-catalytic activity to cyclically cleave the fluorescently quenched ssDNA reporters to recovery drastically amplified fluorescence for detecting MT down to 0.34 nM within the dynamic range of 1∼800 nM. Moreover, the sensing method is able to selectively discriminate MT from other non-specific molecules and can realize low level detection of MT in diluted human serums, manifesting its potentiality for monitoring the disease-specific MT biomarker at trace levels.

A nanozipper structured efficient FRET probe for ratiometric detection of ATP based on target-induced cycling amplification

In the realm of disease diagnostics and prognostics, accurate quantification of adenosine triphosphate (ATP) has become crucial, as the intricate interplay between ATP and cellular metabolism is a fundamental factor in the pathogenesis and progression of many pathological conditions. Herein, a ratiometric fluorescence DNA nanozipper is developed, which enables highly sensitive detection of ATP by employing catalytic hairpin assembly. This innovative approach utilizes target-induced conformational changes in the nanozipper structure to tune the distance between the carboxyfluorescein as donor and tetramethylrhodamine as acceptor around the Förster radius, thereby enhancing the efficiency of fluorescence resonance energy transfer (FRET). In the state of nanozipper tension, the rigid and steric hindrance Y-shaped configuration leads to a considerable distance between two fluorophores, resulting in FRET low efficiency. However, the presence of ATP triggers allosteric configuration of the DNA nanozipper, switching the nanozipper to a relaxed state, which results in a visible fluorescent ratiometric signal. The sensing strategy exhibits a linear response to ATP concentrations in the range of 30 − 600 nM and can detect concentrations as low as 9.6 nM. Additionally, this detection method introduces a novel idea for the design of efficient FRET probes and demonstrates promising application potential in biological samples.

Functional rolling circle amplification-based sensitive determination and low-speed centrifugation-based isolation of Staphylococcus aureus

The ability to quickly and accurately analyze Staphylococcus aureus (S. aureus) and isolate the bacteria in a simplified setting is crucial for the early identification and treatment of infectious illnesses. Here, we describe the development of a new aptamer-based detection and separation technique that combines Mg2+-dependent DNAzyme amplification cascades with catalytic hairpin assembly for enhanced sensitivity. This technique uses a rolling circle amplification procedure to build a detection scaffold with a repetitive functional hairpin structure that, upon identifying S. aureus, can launch a catalytic hairpin assembly-mediated DNAzyme-based cascade signal amplification. This allows S. aureus to be isolated using low-speed centrifugation and simultaneously quantified. The approach has a low limit of detection of 21 cfu/mL and a broad detection range of six orders of magnitude due to the inclusion of the catalytic hairpin assembly for signal amplification. In addition to high sensitivity, the method also demonstrates high selectivity for the identification and isolation of S. aureus, making it a useful instrument for reporting S. aureus infections.

Dual-color nanospheres based on aggregation-induced emission and catalytic hairpin assembly for simultaneous imaging of acrylamide and miR-21 in living cells

Acrylamide (AA) is a heat-processed potent food carcinogen that is widely used in industry, posing a significant risk to human health. Therefore, it is necessary to investigate the toxic effects and mechanism of AA. miR-21 is a representative biomarker during AA-induced carcinogenesis. Here, dual-color aggregation-induced emission nanoparticles (AIENPs) were developed for the detection and simultaneous imaging of AA and miR-21. AIENPs were synthesized by combining aggregation-induced emission (AIE) dyes and a poly (styrene-co-maleic anhydride) (PSMA) amphiphilic polymer modified with hairpin DNA. Upon AA intervention and aptamer recognition, cDNA was dissociated, leading to miR-21 overexpression and initiating the catalytic hairpin assembly cycle. Consequently, fluorescence quenching was observed due to FRET between AIENPs and labeled quenchers. The relative fluorescence intensities of dual-color AIENPs displayed good linear relationships with logarithmic AA and miR-21 concentrations. Moreover, there was a gradual decrease in dual-color AIENP fluorescence as the HepG2 cell concentration of AA (0–500 μM) and stimulation time (0–12 h) increased, making it possible to simultaneously image AA and AA-induced miR-21. The findings of this work are valuable for revealing the cytotoxic mechanism of AA.

APE1 mediated target-responsive structure switching electrochemical (SSE) biosensor for RNA detection

Structure switching caused by the contact between nucleic acids plays an important way for transmit information. Target-responsive structural switching strategy is a promising tool in the development of biosensors. Herein, an apurinic/apyrimidinic endonuclease 1 (APE1) mediated target-responsive Structure Switching Electrochemical (SSE) biosensor was developed for RNA detection. The method is established on that a toehold-mediated hairpin ssDNA, with AP site modified at a specific position, serves as a target capturer and structural transformer and modified on the gold electrode. By target-responsive catalytic hairpin assembly (CHA), the switch of AP sites in double-strand and single-strand is realized. Subsequently, the specifically cleavage of AP site by APE1 achieve the electronical signal output. Both “turn-on” and “turn-off” signal output strategies were design. Under optimal conditions, the SSE biosensor presented wide linear range both in “turn-on” (10 fM to 100 nM) and “turn-off” (1 fM to 100 nM) modes with a LOD as low as 4.5 fM and 0.6 fM respectively. The specificity and reproducibility of the method are satisfactory. To demonstrate the practical applications of this biosensor, we have successfully validated its performance in detecting Strawberry Mottle Virus (SMoV) from strawberry leaf samples. These results highlight the simplicity, high specificity, and sensitivity of this biosensor platform, making it a promising tool for RNA detection.