Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT

MiRNAs hold great potential as biomarkers for the early detection and monitoring of diseases based on their differential expression profiles. Therefore, the sensitive, specific and accurate detection of miRNAs represents an emerging new tool to improve diagnosis and treatment of several diseases, cancer in particular. DNA origami -based miRNA detection is particularly advantageous as it allows to incorporate multiple attachment sites to capture different target miRNAs at the nanoscale. In this work, we present a DNA origami nanoarray system providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA -PAINT (point accumulation for imaging in nanoscale topography). The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. The detection is highly sensitive, with a limit of detection down to the low femtomolar range (11 fM -388 fM) and has a large dynamic range up to 10 nM without need for amplification. Moreover, our detection system can discriminate single base mismatches with low false positive rates. Using our strategy, we demonstrate the detection of endogenous miRNAs from cell extracts of cancer cell lines and plasma from breast cancer patients. Overall, we developed an ultrasensitive and amplification-free, DNA-PAINT imaging-based miRNA detection method using DNA origami nanoarray system for the detection of breast-cancer associated miRNAs which potentially provides a sensitive and accurate alternative to the current multiplexed diagnostic technologies.

Automated summary

miRNA has the potential to be biomarkers for early disease detection, and the sensitive and accurate detection of miRNA can improve diagnosis and treatment of diseases, especially cancer. This study introduces a DNA origami nanoarray system that uses super-resolution microscopy technique DNA-PAINT to detect miRNAs based on their distance to boundary markers. The sensor can detect up to 4 miRNAs separately or in combination, with high sensitivity and large dynamic range, without the need for amplification. The detection system can also discriminate single base mismatches with low false positive rates. This ultrasensitive and amplification-free miRNA detection method has been successfully applied to detect breast-cancer associated miRNAs in cell extracts and plasma.