Barbaria, S. | bioRxiv Science

bioRxiv2024

Snorkel-tag Based Affinity Chromatography for Recombinant Extracellular Vesicle Purification

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine are still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present CD81, an EV surface marker protein, genetically fused to series of tags with additional transmembrane domain to be displayed on the EV surface, which we term Snorkel-tag. This system enables to affinity purify EVs from complex matrices in a non-destructive form. In future applications, this strategy will allow generating transgenic animals to enable tracing and analyzing EVs, and their cargo in physiological and pathophysiological set-ups, and facilitate the development of EV based diagnostic tools in murine models which can be translated to humans.

bioengineering↗

bioRxiv2021

A Bioinformatic and Empiric Exploration of Prokaryotic Argonautes as Novel Programmable Endonuclease Systems

Argonautes are nucleases that can be programmed by short oligonucleotides to cleave complementary sequences. Here, we performed an unbiased bioinformatic search to mine bacterial genomes for prokaryotic Argonautes (pAgos) harboring a PIWI domain. Our search identified 3,033 pAgos in total, of which 1,464 portend to the subgroup of long pAgos with more than 600 amino acids. We purified a subset of 49 pAgos which were found in proximity to helicases and tested their nuclease activity in vitro. Ten of these were active towards single-stranded DNA substrates and this activity could be programmed by exogenous guide DNAs or RNAs. Cleavage of double-stranded plasmid DNA was much less readily observed and was fostered by elevated temperatures or exogenous addition of a DNA single-strand binding protein (ET-SSB). The efficiency of pAgo-mediated plasmid cleavage was dependent on the DNA target sequence as well as the surrounding sequence, suggesting that unwinding of the DNA double helix was a limiting factor. Intriguingly, we identified a cluster of pAgos from the Clostridial clade which was active at 37{degrees}C and activity was enhanced by exogenous ET-SSB. This suggests that Clostridial pAgos may be particularly suited to catalyze DNA double-strand cleavage and implies that such pAgos may be repurposed as gene editing tools in future.

molecular biology↗