Construction of an Autonomous Nonlinear Hybridization Chain Reaction for Extracellular Vesicles-Associated MicroRNAs Discrimination

Abstract

Extracellular vesicles (EVs) have emerged as promising tumor biomarkers for early cancer diagnosis, as primary tumor-secreted EVs carry characteristic molecular information on parent cells. It is thus desirable to realize the efficient discrimination of the signatured EVs-associated microRNAs (miRNAs) with low expression and subtle variation. Here, we introduce an autonomous nonlinear enzyme-free signal amplification paradigm for EVs discrimination through a highly sensitive and selective detection of their inherent miRNAs in situ. Our proposed amplifier consists of a modularized DNAzyme-amplified two-stage cascaded hybridization chain reaction (CHCR–DNAzyme) circuit, where the analyte-generated output of the preceding hybridization chain reaction (HCR1) stage serves as input to motivate the following hybridization chain reaction (HCR2) stage and the concomitant assembly of numerous DNAzyme biocatalysts. By incorporating a flexibly configurable sensing module, this modular CHCR–DNAzyme circuit can further extend to “plug-and-play” sensing mode that enables the miRNA assay with high specificity. The sophisticated design and the detecting performance of our CHCR–DNAzyme scheme were systematically investigated in vitro. The optimized CHCR–DNAzyme system was further applied for distinguishing EVs derived from different cells through the amplified detection of a putative miRNA biomarker in EVs. This compact CHCR–DNAzyme amplifier provides a universal and facile toolbox for highly efficient identification of multiple miRNAs-involved EVs and thus holds great potential for early cancer diagnosis.

Modified in situ Hybridization Chain Reaction Using Short Hairpin DNAs

“…Such advantages were also observed with the short hairpin DNAs designed in this study. Moreover, the current modification for the short hairpin DNA design implies further signal amplification by modifying the hairpin sequence, such as is used in the branched HCR system (Xu and Zheng, 2016;Bi et al, 2017;Liu et al, 2018;Wu et al, 2019).…”

A Smart, Autocatalytic, DNAzyme Biocircuit for in Vivo, Amplified, MicroRNA Imaging

“…[17] Forpromoting the signal amplification of DNAzymes,e fforts have been devoted to its efficient integration with other nonenzymatic amplification approaches,including HCR or CHA systems. [18] Moreover,w eh ave recently reported an intracellular miR-NA-imaging system based on ac oncatenated CHA-HCR-DNAzyme circuit, [19] in which the final DNAzyme layer is utilized merely as af inal transduction element. Yett he amplification capacity of the concatenated system is limited since the DNAzyme is unable to feedback into the previous CHA or HCR system, resulting in inefficient signal amplification.…”

A DNAzyme-amplified DNA circuit for highly accurate microRNA detection and intracellular imaging

“…[42][43][44] And their dysregulated expressions are closely related to cancer development, progression and therapy resistance, 45 which makes them a clinically crucial class of diagnostic and prognostic biomarkers. 46 MiRNA-21(miR-21) has been demonstrated to be an upregulated miRNA in many tumor types. 47,48 Thus the development of highly sensitive miRNA sensing platforms represents an urgent requirement for detecting low abundance miRNAs without elaborate separation and enrichment processes in the complex intracellular environment.…”