Selective In Situ Analysis of Mature microRNAs in Extracellular Vesicles Using a DNA Cage‐Based Thermophoretic Assay

Abstract

Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre‐miRNAs) and the low abundance of tumor‐associated miRNAs. By leveraging the size‐selective ability of DNA cages and polyethylene glycol (PEG)‐enhanced thermophoretic accumulation of EVs, we devised a DNA cage‐based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05 fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre‐miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR‐21 or miR‐155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre‐miRNAs. We envision that our assay can advance EV miRNA‐based diagnosis of cancer.

Smart DNA sensors‐based molecular identification for cancer subtyping

“…Nevertheless, most nanosensors easily degrade in serum and the pre-miRNAs (longer precursor miRNAs) containing identical sequences in EVs remained an issue to precisely recognize mature miRNAs. To meet these challenges, Sun et al [45] devised DNA cages that have considerable selectivity of size and stability in thermophoresis for the detection of mature miRNAs in EVs (Figure 6b). With this design, the sensor can detect mature miRNAs at a LOD of 2.05 fM without the interference of pre-miRNAs, which is comparable to the results of gold standard RT-PCR.…”

“…[44] (b) The thermophoretic implemented with size-selective DNA cage for detection of mature miRNAs in EVs. [45] (c) Thermophoresis-mediated DNA computation approach for detection of receptors on EV membranes. [46] (d) Homotypic recognition-driven molecular characterization of BC exosomes (EXOs) based on using camouflaging catalytic DNA machinery.…”

“…[46] (d) Homotypic recognition-driven molecular characterization of BC exosomes (EXOs) based on using camouflaging catalytic DNA machinery. [47] Graph copyright from reference [44][45][46][47] . associated with this.…”

Intracellular MicroRNA Imaging and Specific Discrimination of Prostate Cancer Circulating Tumor Cells Using Multifunctional Gold Nanoprobe-Based Thermophoretic Assay

“…Aiming to improve the sensitivity of miRNAs detection, various signal amplification systems have been developed, including nanomaterial-mediated amplification procedures [e.g., gold nanoparticles (GNPs), graphene nanosheets, and so on] − and molecular biological technique-based amplification strategies (e.g., hybridization chain reaction, strand-displacement amplification, and other DNA nanotechnology-based methods). − Among them, GNPs with excellent biocompatibility, facile preparation, and easy modification have been widely used in constructing signal amplification systems. ,,− Thermophoresis as an effective signal amplification approach utilizes the responses of particles to temperature gradient, thus inducing the motion and aggregation of particles, and finally achieving the signal amplification . Taking advantage of label-free and the capability of selectively driving suspended objects into warm or cold regions along a temperature gradient, thermophoresis has been employed for miRNA detection. , For example, Sun’s group reports a thermophoretic sensor for in situ detection of exosomal miRNAs with high accuracy, which relies on nanoflare detection of miRNAs and thermophoretic enrichment of cancer cell-derived exosomes . Therefore, we rationally designed a multifunctional gold nanoprobe for simultaneous specific distinguishment of prostate cancer CTCs and sensitive imaging of upregulated miR-21 in CTCs through thermophoresis.…”

“…46 Taking advantage of label-free and the capability of selectively driving suspended objects into warm or cold regions along a temperature gradient, thermophoresis has been employed for miRNA detection. 47,48 For example, Sun's group reports a thermophoretic sensor for in situ detection of exosomal miRNAs with high accuracy, which relies on nanoflare detection of miRNAs and thermophoretic enrichment of cancer cell-derived exosomes. 47 Therefore, we rationally designed a multifunc- In this work, a multifunctional gold nanoprobe-based thermophoretic assay was reported for simultaneously monitoring aberrant miR-21 expression in CTCs and visually discriminating CTCs, achieving the diagnosis of prostate cancer.…”