Development of DNA Aptamers to Visualize Release of Mycobacterial Membrane-Derived Extracellular Vesicles in Infected Macrophages

Abstract

Extracellular vesicles (EVs) have emerged into a novel vaccine platform, a biomarker and a nano-carrier for approved drugs. Their accurate detection and visualization are central to their utility in varied biomedical fields. Owing to the limitations of fluorescent dyes and antibodies, here, we describe DNA aptamer as a promising tool for visualizing mycobacterial EVs in vitro. Employing SELEX from a large DNA aptamer library, we identified a best-performing aptamer that is highly specific and binds at nanomolar affinity to EVs derived from three diverse mycobacterial strains (pathogenic, attenuated and avirulent). Confocal microscopy revealed that this aptamer was not only bound to in vitro-enriched mycobacterial EVs but also detected EVs that were internalized by THP-1 macrophages and released by infecting mycobacteria. To the best of our knowledge, this is the first study that detects EVs released by mycobacteria during infection in host macrophages. Within 4 h, most released mycobacterial EVs spread to other parts of the host cell. We predict that this tool will soon hold huge potential in not only delineating mycobacterial EVs-driven pathogenic functions but also in harboring immense propensity to act as a non-invasive diagnostic tool against tuberculosis in general, and extra-pulmonary tuberculosis in particular.

DNA Aptamer Targets Mycobacterium tuberculosis DevR/DosR Response Regulator Function by Inhibiting Its Dimerization and DNA Binding Activity

“…However, in eukaryotes, aptamers are taken into cells typically by clathrin-mediated endocytosis or micropinocytosis followed by their transport to various cell organelles . Recently, a DNA aptamer targeting mycobacterial extracellular vesicles (mEVs) was visualized to bind mEVs inside the cytosol of THP-1 macrophages, providing evidence for the aptamer’s ability to traverse intracellular membranes. Additionally, DNA aptamers exhibiting high affinity and selectivity for cytokine-stimulated cells were shown to colocalize stably with LAMP-1, a late endosomal and lysosomal marker, suggesting that endosomal or lysosomal pH does not affect aptamer function inside host cells .…”

Polymers in Engineering Extracellular Vesicle Mimetics: Current Status and Prospective

“…Natural polymers, such as proteins and aptamers, have been widely used to better imitate [ 133 , 134 ] and optimize targeting functions [ 135 , 136 ]. Synthetic polymers such as PEG, PEI, and pCBMA can be utilized for more efficient delivery [ 91 , 116 , 117 , 137 ].…”

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell‐Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent

“…[34][35][36][37][38] Furthermore, the aptamers obtained via SELEX have been used for biomarker discovery and validation, thereby identifying potential biomarkers for diagnostics and therapeutics. [39][40][41] Despite the emerging clinical significance of sEVs, only a few studies employing SELEX with the use of sEVs originated from mammalian and bacterial sources as the target have been reported, [42][43][44] emphasizing high demand for the development of aptamers that are specific to sEVs.…”