Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Diagnostics

Background:

• MicroRNAs (miRNAs) are vital biomarkers for disease detection, particularly cancer, due to their differential expression.
• Accurate and sensitive detection of miRNAs is essential for improving disease diagnosis and treatment strategies.
• Current diagnostic technologies require enhancement for sensitive and specific miRNA detection.

Purpose of the Study:

• To develop an ultrasensitive, amplification-free method for detecting multiple microRNAs (miRNAs) simultaneously.
• To utilize DNA origami nanoarrays and super-resolution microscopy (DNA-PAINT) for distance-dependent miRNA recognition.
• To demonstrate the application of this system for detecting cancer-associated miRNAs in biological samples.

Main Methods:

• Fabrication of a DNA origami nanoarray with multiple capture sites for target miRNAs.
• Application of DNA-PAINT super-resolution microscopy for nanoscale imaging and distance-dependent recognition.
• Detection of up to four miRNAs using a single imager strand and boundary markers.

Main Results:

• Achieved a limit of detection in the low femtomolar range (11–388 fM) with a broad dynamic range up to 10 nM.
• Demonstrated amplification-free detection with high sensitivity and specificity.
• Successfully detected endogenous miRNAs from cancer cell line extracts and breast cancer patient plasma.
• Showcased the ability to discriminate single-base mismatches, ensuring low false positive rates.

Conclusions:

• Developed an ultrasensitive, amplification-free miRNA detection method using DNA origami and DNA-PAINT.
• The system offers a sensitive and accurate alternative for multiplexed diagnostic technologies.
• This approach holds potential for early cancer detection and disease monitoring.