Dataset Information

Intracellular MicroRNA imaging using telomerase-catalyzed FRET ratioflares with signal amplification.

ABSTRACT: Intracellular microRNA (miRNA) detection has attracted increasing attention, resulting in significant achievements. However, the development of an available tool that possesses a satisfactory signal-to-background ratio and high sensitivity for miRNA detection remains challenging. Herein, a class of telomerase-catalyzed FRET (fluorescence resonance energy transfer) ratioflares has been developed for the accurate sensing of low-abundance cancer-related miRNA both in a fluorescence assay and living cell imaging with signal amplification capacity. In this work, endogenous telomerase is led in a miRNA test system with signal amplification for the first time, wherein telomerase extends hexamer telomeric repeats (TTAGGG) using the 3' end of the capture probe as the primer. The synergetic work of telomerase and the catalyst strand (CS) makes the target miRNA circulate in the system, resulting in high sensitivity and an enhanced FRET signal an improved detection limit of 2.27 × 10-15 M. Meanwhile, telomerase-catalyzed FRET ratioflares allow the difference between cancer cells and normal cells to be increased reliably. Furthermore, low false positive signals resulting from chemical interference and minimized system fluctuations are achieved through ratiometric measurements.

SUBMITTER: Xian L

PROVIDER: S-EPMC6677110 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Intracellular MicroRNA imaging using telomerase-catalyzed FRET ratioflares with signal amplification.

Xian Liman L Ge Haoying H Xu Feng F Xu Ning N Fan Jiangli J Shao Kun K Peng Xiaojun X

Chemical science 20190607 29

Intracellular microRNA (miRNA) detection has attracted increasing attention, resulting in significant achievements. However, the development of an available tool that possesses a satisfactory signal-to-background ratio and high sensitivity for miRNA detection remains challenging. Herein, a class of telomerase-catalyzed FRET (fluorescence resonance energy transfer) ratioflares has been developed for the accurate sensing of low-abundance cancer-related miRNA both in a fluorescence assay and living ...[more]

PMID: 31588279

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Project description:Biomolecular self-assembly has spurred substantial research efforts for the development of low-cost point-of-care diagnostics. Herein, we introduce an isothermal enzyme-free concatenated hybridization chain reaction (C-HCR), in which the output of the upstream hybridization chain reaction (HCR-1) layer acts as an intermediate input to activate the downstream hybridization chain reaction (HCR-2) layer. The initiator motivates HCR-1 through the autonomous cross-opening of two functional DNA hairpins, yielding polymeric dsDNA nanowires composed of numerous tandem triggers T as output of the primary sensing event. The reconstituted amplicon T then initiates HCR-2 and transduces the analyte recognition into an amplified readout, originating from the synergistic effect between HCR-1 and HCR-2 layers. Native gel electrophoresis, atom force microscopy (AFM) and fluorescence spectra revealed that C-HCR mediated the formation of frond-like branched dsDNA nanowires and the generation of an amplified FRET signal. As a versatile and robust amplification strategy, the unpreceded C-HCR can discriminate DNA analyte from its mutants with high accuracy and specificity. By incorporating an auxiliary sensing module, the integrated C-HCR amplifier was further adapted for highly sensitive and selective detection of microRNA (miRNA), as a result of the hierarchical and sequential hybridization chain reactions, in human serum and even living cells through an easy-to-integrate "plug-and-play" procedure. In addition, the C-HCR amplifier was successfully implemented for intracellular miRNA imaging by acquiring an accurate and precise signal localization inside living cells, which was especially suitable for the ex situ and in situ amplified detection of trace amounts of analyte. The C-HCR amplification provides a comprehensive and smart toolbox for highly sensitive detection of various biomarkers and thus should hold great promise in clinical diagnosis and assessment. The infinite layer of multilayered C-HCR is anticipated to further strengthen the amplification capacity and reliability (anti-invasion performance) of intracellular imaging approach, which is of great significance for its bioanalytical applications.

Dataset Information

Intracellular MicroRNA imaging using telomerase-catalyzed FRET ratioflares with signal amplification.

Publications

Intracellular MicroRNA imaging using telomerase-catalyzed FRET ratioflares with signal amplification.

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