Simultaneous Imaging of Multiple miRNAs in Mitochondria Controlled by Fluorescently Encoded Upconversion Optical Switches for Drug Resistance Studies

Abstract

Mitochondrial miRNAs (mitomiRs) are essential regulators of biological processes by influencing mitochondrial gene expression and function. To comprehensively understand related pathological processes and treatments, simultaneous imaging of multiple mitomiRs is crucial. In this study, we present a technique that enables simultaneous monitoring of multiple mitomiRs in living cells using a near-infrared (NIR) photoactivated controlled detection probe (PD-mFleU) with a fluorescence-encoded error correction module and a nonsupervised machine learning data-processing algorithm. This method allows controlled sensing imaging of mitomiRs with a DNA reporter probe that can be activated by NIR light after targeted mitochondrial localization. Multilayer upconversion nanoparticles (UCNPs) are used for encoding probes and error correction. Additionally, the density-based spatial clustering of applications with the noise (DBSCAN) algorithm is used to process and analyze the image. Using this technique, we achieved rapid in situ imaging of the abnormal expression of three mitomiRs (miR-149, miR-590, and miR-671) related to mt-ND1 in drug-resistant cells. Furthermore, upregulating the three mitomiRs simultaneously efficiently reverted drug-resistant cells to sensitive cells. Our study provides an analytical strategy for multiplex imaging of mitomiRs in living cells with potential clinical applications.

Fluorescence coding techniques for RNA detection

“…Each barcode in the sensor simultaneously packaged the same number of AMCA fluorophores for error correction. 116 Finally, they acquired in situ pictures of the three separate miRNAs. Here, C represents the number of distinguishable spectral features ('colors') and S represents the number of spatial features ('stripes').…”

“…Each barcode in the sensor simultaneously packaged the same number of AMCA fluorophores for error correction. 116 Finally, they acquired in situ pictures of the three separate miRNAs.…”

Remotely Activated DNA Probe System for the Detection and Imaging of Dual miRNAs

“…Given that single miRNA detection could not provide enough information, multiplexed detection of biomarkers was accomplished through the integration of DNA frameworks by targeting the vertex of a three-dimensional (3D) DNA structure , or by employing microRNA as a trigger to initiate multiple amplification processes. − To achieve simultaneous imaging, multiple DNA fluorescent probes were reported to be embedded in the outer layer of UCNP . Although a number of DNA-based probes for the in vivo imaging of multiple miRNAs have been described, more strategies that are capable of recognizing and capturing multiple biomarkers are still urgently needed.…”