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In this work, we propose a hairpin probe-mediated exponential amplification reaction (HEAR) strategy that combines DNA strand displacement with a "who triggers, who gets generated" mode, providing excellent single-base discrimination and a reduced background signal. The detection limit is 19 aM, which is reduced by 3 orders of magnitude compared to traditional exponential amplification approaches. This one-pot strategy also exhibits a wide dynamic range, high specificity and short detection time. It is expected to become a powerful tool for clinical diagnosis. [ABSTRACT FROM AUTHOR]

Nucleic acid hybridization plays a critical role in medical diagnostics and nanotechnology, but its selectivity and robustness remain to be improved. Here, focusing on double-stranded nucleic acid-based hybridization, we present a series of related strategies. Above all, two simple strategies for enriching toehold-included double-stranded nucleic acids have been proposed. On this basis, two universal hybridization methods with higher selectivity than typical toehold exchange reaction and a long target detection method using short probes to extend the detectable length range are realized. We also provide a double-stranded nucleic acids-catalyzed cycle amplification reaction to improve sensitivity, which has superior interference resistance and excellent discrimination for single-base mismatches. Besides, double-stranded nucleic acids with forked toeholds are used as essential elements to construct a series of logic gates that can evaluate different input combinations. Given the unique advantages of double-stranded nucleic acids, we expect the current work to advance the application of double-stranded nucleic acid-based hybridization in medical diagnostics and nanotechnology. [Display omitted] • Two methods for enriching double-stranded nucleic acids that possess toeholds. • Two universal hybridization methods and a long targets detection method were provided. • A double-stranded nucleic acids-catalyzed cycle amplification reaction for improving sensitivity. • Double-stranded nucleic acids for robust logic computation. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in RNA and has important implications in physiological processes and tumor development. However, sensitive and specific quantification of locus-specific m6A modification levels remains a challenging task. In the present work, a novel m6A-sensitive DNAzyme was utilized to directly detect m6A by coupling with a three-way junction-mediated isothermal exponential CRISPR amplification reaction for the first time. This method was built on the fact that the binding arm of the DNAzyme bound to the specific site and its core structure catalyzed the selective cleavage of unmodified adenine instead of methylated adenines. Subsequently, the intact RNA was identified by the proximity effect of the three-way junction. Enormous amounts of single-stranded DNA products were generated through a combination of SDA and EXPAR for signal amplification. The specific real-time curve of products was recorded through detecting the fluorescence intensity triggered by CRISPR Cas12a. As a result, methylation target of abundance down to 1% was successfully identified. In addition, this strategy could be used for the analysis of cell RNA extracts. Combined with an electrochemical sensor for quantitative detection of RNA methylation, we demonstrated the generality of as-proposed strategy. We envision the present method would provide a new platform for the analysis of m6A in RNA and promote its application in clinical diseases. • DNAzyme and the three-way junction were firstly used to specifically identify m6A RNA. • The integration of EXPAR and CRISPR Cas12a assisted exponential amplification realized sensitive detection of m6A modifications down to 1% abundance. • This strategy was easily elaborated into multiple reactions for different detection purposes. [ABSTRACT FROM AUTHOR]

In this study, an ultrasensitive electrochemical miRNA biosensor based on toehold mediated strand displacement reaction circuits (SDRCs) and molecular beacon mediated isothermal circular strand displacement polymerization reaction (ICSDPR) has been proposed. During the SDRCs module, the cascade strand displacement reaction induces the recycling of the target let 7a and generation of a large amount of strand A (SA). The SA recognition opens the hairpin capture probe immobilized on the gold electrode, thus, varying the distance between the redox molecules and electrode surface. The primer mediated ICSDPR is observed to further generate a large amount of SA, thus, leading to a reduction in the signal. Considering these merits, the proposed method is observed to exhibit a log-linear linearity from 10 aM to 100 pM and ultrahigh sensitivity towards let 7a down to 6.2 aM, with a capability of distinguishing the let 7a family members, thereby, providing a new electrochemical route for early cancer screening. Isothermal amplification combined SDRCs and ICSDPR for sensitive and selective let 7a detection. Image 1 • A multiple amplification strategy based on SDRCs and ICSDPR was designed. • The SDRCs and ICSDPR process exhibited good selectivity and amplified the signal efficiently. • The method exhibited high sensitivity with the LOD of 6.2 aM for miRNA let 7a. • The proposed strategy achieved extensive prospect in complex matrix and real samples of cells. [ABSTRACT FROM AUTHOR]

Sensitive and specific miRNA detection is essential for the early cancer diagnosis. In this work, we design a fluorescent microRNA biosensor based on exponential amplification reaction (EXPAR) and nicking endonuclease-powered three-dimensional (3-D) bipedal DNA walkers (BDW). Target microRNA initiates EXPAR with the help of polymerase and nicking endonuclease to generate the large number of BDW in solution. The newly generated BDW can be continuously assembled onto polystyrene microsphere track co-modified with fluorescence-labeled DNA strand. Thus, in the presence of nicking endonuclease, the walking machine is activated to produce enhanced fluorescent signal in the supernatant. Besides, we prove that BDW holds the faster walking speed than single-legged DNA walker (SDW) based on comparative study. Under optimal conditions, the proposed amplification method owns a wide linear range from 10 fM to 5 nM with a detection limit down to 5.2 fM. The reaction time of the assay takes about 70 min. The combination of enzyme-assisted EXPAR in solution and enzyme-powered BDW on particle significantly increases the signal amplification efficiency and improves the detection sensitivity. Therefore, our method has enormous potential for the application of BDW-related biosensors. EXPAR-BDW strategy based on exponential amplification reaction-triggered three-dimensional bipedal DNA walkers. Image 1 • A strategy for detection microRNA was constructed based on exponential amplification reaction and 3-D bipedal DNA walkers. • The proposed assay shows high sensitivity towards target microRNA 21 with a detection limit of 5.2 fM. • This method is simple in nucleic acid design and can be completed in short measurement time. [ABSTRACT FROM AUTHOR]