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Comment
. 2022 Sep;12(9):e1059.
doi: 10.1002/ctm2.1059.

Short prokaryotic Argonaute system repurposed as a nucleic acid detection tool

Ana Potocnik  1 , Daan C Swarts  1
Affiliations
Comment

Short prokaryotic Argonaute system repurposed as a nucleic acid detection tool

Ana Potocnik et al. Clin Transl Med. 2022 Sep.
No abstract available

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Conflict of interest statement

Ana Potocnik and Daan C. Swarts (together with Balwina Koopal) submitted a patent application regarding the utilization of short pAgo systems for nucleic acid detection.

Figures

FIGURE 1
FIGURE 1
Short prokaryotic Argonaute/TIR‐APAZ (SPARTA) is a bacterial immune system that provides its bacterial host with population‐level immunity against invading plasmids. SPARTA acquires guide RNAs from transcripts of plasmid‐encoded genes to bind complementary plasmid DNA targets. Upon target DNA binding, SPARTA oligomerizes and becomes catalytically activated, after which its NAD(P)ase is unleashed, depleting the cell of NAD(P)+. This triggers cell death in plasmid‐invaded cells, removing plasmid‐invaded cells from the bacterial culture
FIGURE 2
FIGURE 2
Schematic representation of the Short prokaryotic Argonaute/TIR‐APAZ (SPARTA)‐based nucleic acid detection tool. Left panel: SPARTA can be isolated and re‐programmed with guide RNAs of choice to detect DNA in vitro. Top panel: SPARTA‐based ssDNA detection. SPARTA can be provided with a synthetic RNA guide with a sequence of choice. This allows the detection of complementary single‐stranded DNA target sequences in pools of DNA. The presence of specific DNA sequences can be detected by supplementing the reaction with etheno‐NAD (ɛ‐NAD), an analogue of NAD+ that is converted into fluorescent ɛ‐ADPR by activated SPARTA complexes. Bottom panel: SPARTA‐based dsDNA detection. To enable dsDNA detection and to increase the sensitivity of SPARTA‐based detection, target DNA can be specifically amplified by PCR using a phosphorothioate (PT) forward primer and an unmodified reverse primer. Upon incubation with T7 exonuclease, the unmodified strand is degraded, leaving ssDNA fragments containing the target sequence that can be detected by SPARTA
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