Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level

Zhou, Xueqing; Liang, Yao; Xu, Yuzhi; Lin, Xiulian; Ma, Yingjun; Zhang, Li; Chen, Danping; Song, Feifei; Dai, Zong; Zou, Xiaoyong

doi:10.1016/j.bios.2016.01.059

Cited by 11 publications

(3 citation statements)

References 31 publications

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“…To verify the feasibility of the proposed method, microRNA (miRNA) was selected as a proof-of-concept target. MiRNAs are a group of endogenous noncoding RNAs with a length of approximately 22 nucleotides that play important roles in numerous biological processes such as cell development, differentiation, and immune system. − The miRNAs expression levels are associated with various pathological conditions, which are commonly used as important diagnostic and prognostic biomarkers. − However, the intrinsic characteristics of miRNAs, including low abundances, easy degradations, small sizes, and sequence similarities among family members, make developing detection techniques with ultrahigh specificities and sensitivities intensely challenging. , Conventional analytical methods, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), , Northern blotting, microarray, and surface plasmon resonance (SPR), are widely utilized for miRNA analysis. However, these miRNA detection techniques are complicated, expensive, and time-consuming.…”

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confidence: 99%

An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity

et al. 2017

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Graphene oxide (GO) has been proven as an outstanding fluorescence anisotropy (FA) amplifier. Yet the traditional GO amplified FA assays lack high sensitivity because of the 1:1 binding ratio between target and dye-modified probe. Herein, we report a new target-catalyzed hairpin assembly (CHA), an enzyme-free DNA circuit, assisted GO amplified FA strategy for microRNA-21 (miRNA-21) detection. In the presence of miRNA-21, the CHA was initiated and plenty of H1-H2 duplexes were produced continuously. The obtained H1-H2 duplex could induce the formation of a H1-H2-probe DNA (pDNA) complex by the toehold-mediated strand exchange reaction, which led the dye-modified pDNA to leave away from the GO surface, resulting in a decreased FA of the system. By monitoring the decrease of FA, miRNA-21 could be detected in the range of 0-16 nM. The limit of detection (LOD, 3σ) was 47 pM, which was 194 times lower than that without CHA. In addition, the selectivity of this method has also been enhanced greatly as compared to that without CHA. Our method has great potential to be applied for detecting different types of targets and monitoring diverse molecular interactions by adapting the corresponding nucleotide sequence.

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confidence: 99%

An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity

et al. 2017

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“…While the sensing of biomolecules, and of nucleic acids in particular, via isothermal signal amplification has been of intense recent interest, − few examples exist for the sensing of proteins via purely DNA-based enzyme-free methodologies. Many sensing schemes require polymerase, nicking enzymes, or the incorporation of nanoparticle or bead-based approaches.…”

Section: Resultsmentioning

confidence: 99%

Combining Affinity Selection and Specific Ion Mobility for Microchip Protein Sensing

et al. 2018

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The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by nonspecific interactions with off-target species. Here, we describe and demonstrate an assay that utilizes both the chemical and physical properties of the target species to achieve high selectivity in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nanocircuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilization or washing steps are required, and our assay is well suited to monoepitopic targets, presenting advantages over conventional ELISA techniques.

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“…Thus, there is interest in studying different aspects of isomiRs, such as their up-/down-regulation or interactions with other biomolecules, and this necessitates the accurate and efficient detection of different, specific isomiRs [ 6 , 7 ]. Due to low miRNA copy numbers, analysis of miRNA is often focused on improving the sensitivity of detection and as a result modes of miRNA amplification such as enzyme cascades, real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification and rolling cycle amplification [ 8 – 10 ] have been employed. These detection methods almost always involve an initial hybridization step by probes/primers.…”

Section: Introductionmentioning

confidence: 99%

Hi-fidelity discrimination of isomiRs using G-quadruplex gatekeepers

et al. 2017

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Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.

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Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level

Cited by 11 publications

References 31 publications

An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity

An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity

Combining Affinity Selection and Specific Ion Mobility for Microchip Protein Sensing

Hi-fidelity discrimination of isomiRs using G-quadruplex gatekeepers

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