DNA Zipper Mediated Membrane Fusion for Rapid Exosomal MiRNA Detection

Abstract

Accurate and reliable detection of exosomal miRNA can serve as a promising method for early diagnosis of disease and evaluation of therapeutic effects. However, current exosomal miRNA detection methods commonly involve exosome enrichment, containing RNA extraction, and qRT-PCR based quantification, which are expensive and time-consuming. Herein, we develop a DNA zipper-mediated membrane fusion approach for rapid exosomal miRNA detection and cancer diagnosis. First, a lipid vesicle probe containing miR21-targeting molecular beacons (MBs) is constructed and further loaded with zipper DNA constructs (ZDCs) on its surface. Meanwhile, complementary zipper DNA constructs (cZDCs) are introduced on the exosome of interest. Upon mixing them together, zipping between ZDC and cZDC induces the membrane fusion of exosomes and vesicle probes, triggering the recognition of exosomal miR21 by contained MBs and fluorescence emission that can be conveniently detected within 30 min. Importantly, with the assistance of flow cytometry, miR21-overexpressed tumor exosomes derived from either cell culture medium or clinical patient serums can be distinguished from exosomes secreted from normal cells. This approach provides a convenient way to accurately detect the exosomal miRNA, which may hold great potential in liquid biopsy for early cancer diagnosis and monitoring the therapeutic effects during the treatments.

Quantifying DNA-mediated liposome fusion kinetics with a fluidic trap

“…Combined with the chip format this has potential applications in the development of engineered exosomes for drug delivery schemes, where exosomes are fused with liposomes. 13,40…”

“…12 This provides a route to engineered exosomes with potential applications in diagnostics, drug delivery and synthetic biology. 13…”

“…12 This provides a route to engineered exosomes with potential applications in diagnostics, drug delivery and synthetic biology. 13 Despite the development of single-vesicle imaging assays with vesicles tethered to a surface, 1,14 most experiments are still done in bulk as tethering may affect the fusion process. In bulk experiments, two homogeneous sub-populations of synthetic vesicles equipped with complementary biomolecules are prepared at high concentrations and characterized prior to fusion in a separate experiment.…”

Programmable Nanostructures Based on Framework-DNA for Applications in Biosensing

“…Traditional assays include exosome isolation [ 69 ] and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, which is often expensive, laborious, and experimentally challenging [ 70 , 71 ]. A DNA zipper-mediated membrane fusion method for rapid detection of exosome miRNAs can effectively solve the challenges of high cost and time consumption in traditional exosome miRNA detection [ 72 ]. Different from the above detection in homogeneous solutions, a highly ordered 3D DNA nanomachine assembled from an azobenzene-functionalized DNA clamp has been developed in solid-state sensors.…”

DNA Nanomaterial‐Based Optical Probes for Exosomal miRNA Detection

“…The engineered membranes and the fusion of vesicles by the action of membrane proteins greatly improved the delivery efficiency of MB into exosomes. The rapid fusion of vesicles with exosomes was able to avoid time‐consuming pre‐isolation procedures and also prevent exosome damage [23] . In addition, in‐situ detection of exosomal miRNA by fusing vesicles efficiently prevented the degradation and contamination of samples.…”