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Updated: Sep 21, 2026
MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method
Published on: October 7, 2025
A double-key responsive TDNs-HC/Cas13a DNA circuit for free-amplified detection and precise imaging of miRNAs in
Shixiu Xiao1, Yuying Liang2, Yihan Zhang2
1Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), Guangxi Key Laboratory of Chemistry and Molecular Engineering of Medicinal Resources, University Engineering Research Center for Chemistry of Characteristic Medicinal Resources (Guangxi), School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin, 541004, China; School of Medicine, Guangxi University of Science and Technology, Liuzhou, 545005, China.
Background:
The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated (Cas) proteins is an RNA-guided gene editing system with high targeting specificity. Its exceptional recognition capability for target genes has demonstrated immense potential in the field of biosensing. However, the effective integration and delivery of CRISPR/Cas systems and nucleic acid hybridization for precise imaging and detection of lowly expressed analyte in cellulo remains a critical challenge. Here, an integrated TDNs-HC/Cas13a DNA circuit was constructed for free-amplified detection and precise imaging of miRNAs in living cell. The novel design of this method utilizes DNA Tetrahedrons (TDNs) as nanoscaffolds, with three vertices assembled to incorporate miRNA-155-responsive CRISPR/Cas13a and the lock that recognizes miRNA-21, enabling precise molecular recognition through an AND logic gate mechanism.
Results:
This TDNs-HC/Cas13a strategy integrated target recognition module, logical operations module, and signal output module, enabling intracellular co-delivery of elements of module without external vectors. The dual target recognition and synergistically signal-amplification of CRISPR/Cas13a enabled the sensitive and free-amplified detection of miRNA-155 and miRNA-21, and the limit of detection is 32 pM and 5 pM, respectively. At the same time it can be applied for the expression level analysis and single-cell imaging of miRNA-155 and miRNA-21 in cells. Experimental results show that the TDNs-HC/Cas13a system effectively discriminates between normal cells and cancer cells based on fluorescence intensity, confirming its capability for specific imaging of cancer cells.
Significance And Novelty:
This design likes a dual-password safe lock, precisely excluding other cells that express only a single marker or ingest a small number of probe molecules, significantly improving the signal-to-noise ratio and accuracy of detection and imaging.
