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Construction of an enzyme-free concatenated DNA circuit for signal amplification and intracellular imaging.

ABSTRACT: Nucleic acid circuits have shown promising potential for amplified detection of biomarkers with interest in biologically important engineering applications. In this work, by properly integrating two signal amplification approaches, catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), a concatenated CHA-HCR system was established as an isothermal enzyme-free amplification strategy for highly sensitive and selective nucleic acid assay. The target catalyzes the self-assembly of CHA hairpin substrates into dsDNA products, where the split segments of HCR trigger are successively connected to drive the subsequent autonomous cross-opening of HCR hairpins, leading to the construction of HCR tandem copolymeric dsDNA nanowires. The resulting HCR copolymer brings a fluorophore donor/acceptor pair into close proximity that allows an efficient generation of FRET readout signal. Moreover, the optimized CHA-HCR circuit, upon the incorporation of an auxiliary sensing module, can be converted into a universal sensing platform for detecting cancerous biomarkers (e.g., a well-known oncogene miR-21) through a convenient easy-to-integrate procedure. The concatenated CHA-HCR amplifier enables accurate intracellular miRNA imaging in living cells, which is especially suitable for in situ amplified detection of lowly expressed endogenous analytes. The inherent synergistically accelerated recognition and hybridization features of CHA-HCR circuit contribute to the amplified detection of endogenous RNAs in living cells. The flexible and programmable nature of the homogeneous CHA-HCR system provides a versatile and robust toolbox for a wide range of research fields, such as in vivo bioimaging, clinical diagnosis and environmental monitoring.

SUBMITTER: Wang H

PROVIDER: S-EPMC6050587 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Construction of an enzyme-free concatenated DNA circuit for signal amplification and intracellular imaging.

Wang Huimin H Li Chunxiao C Liu Xiaoqing X Zhou Xiang X Wang Fuan F

Chemical science 20180606 26

Nucleic acid circuits have shown promising potential for amplified detection of biomarkers with interest in biologically important engineering applications. In this work, by properly integrating two signal amplification approaches, catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), a concatenated CHA-HCR system was established as an isothermal enzyme-free amplification strategy for highly sensitive and selective nucleic acid assay. The target catalyzes the self-assembly of ...[more]

PMID: 30079197

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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

Construction of an enzyme-free concatenated DNA circuit for signal amplification and intracellular imaging.

Publications

Construction of an enzyme-free concatenated DNA circuit for signal amplification and intracellular imaging.

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