Spatial confinement-enhanced CHA system based on tetrahedral DNA nanostructures for sensitive in situ mRNA analysis

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nanotechnology

Background:

• Accurate detection of tumor-related mRNA is crucial for early cancer diagnosis but faces challenges like low biomarker abundance.
• Existing methods often lack the sensitivity and efficiency required for reliable early cancer detection.

Purpose of the Study:

• To develop a sensitive and efficient platform for detecting tumor-related mRNA, specifically UBE2C mRNA in hepatocellular carcinoma (HCC).
• To demonstrate the modular adaptability of the platform for detecting other RNA targets, such as microRNA-21 (miR-21).

Main Methods:

• Development of a three-dimensional DNA nanomachine (T-CHA) integrating catalytic hairpin assembly (CHA) and a tetrahedral DNA nanostructure (TDN).
• Utilizing T-CHA to trigger localized, cascading amplification reactions for target mRNA detection.
• Application of T-CHA to clinical tissue sections for visualizing gene expression at single-cell resolution.

Main Results:

• T-CHA achieved quantitative detection of UBE2C mRNA with a limit of detection of 8.53 fM within 30 minutes.
• The platform accurately distinguished HCC cells based on UBE2C expression levels.
• T-CHA demonstrated superior performance compared to fluorescence in situ hybridization (FISH) in visualizing heterogeneous UBE2C expression.
• T-CHA successfully detected miR-21, showcasing its adaptability for diverse RNA targets.

Conclusions:

• The T-CHA platform combines structural precision of TDN with signal amplification of CHA for robust and precise molecular diagnosis of HCC.
• T-CHA shows significant potential for early cancer diagnosis and molecular subtyping.
• The modular design of T-CHA supports its application for detecting various RNA biomarkers, paving the way for broader diagnostic applications.