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Updated: Jun 23, 2025
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
Nanopores Reveal the Stoichiometry of Single Oligoadenylates Produced by Type III CRISPR-Cas
David Fuentenebro Navas1, Jurre A Steens2, Carlos de Lannoy3,4
1Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708WE Wageningen, The Netherlands.
Abstract:
Cyclic oligoadenylates (cOAs) are small second messenger molecules produced by the type III CRISPR-Cas system as part of the prokaryotic immune response. The role of cOAs is to allosterically activate downstream effector proteins that induce dormancy or cell death, and thus abort viral spread through the population. Interestingly, different type III systems have been reported to utilize different cOA stoichiometries (with 3 to 6 adenylate monophosphates). However, so far, their characterization has only been possible in bulk and with sophisticated equipment, while a portable assay with single-molecule resolution has been lacking. Here, we demonstrate the label-free detection of single cOA molecules using a simple protein nanopore assay. It sensitively identifies the stoichiometry of individual cOA molecules and their mixtures from synthetic and enzymatic origin. To achieve this, we trained a convolutional neural network (CNN) and validated it with a series of experiments on mono- and polydisperse cOA samples. Ultimately, we determined the stoichiometric composition of cOAs produced enzymatically by the CRISPR type III-A and III-B variants of Thermus thermophilus and confirmed the results by liquid chromatography-mass spectroscopy (LC-MS). Interestingly, both variants produce cOAs of nearly identical composition (within experimental uncertainties), and we discuss the biological implications of this finding. The presented nanopore-CNN workflow with single cOA resolution can be adapted to many other signaling molecules (including eukaryotic ones), and it may be integrated into portable handheld devices with potential point-of-care applications.
Insights
We developed a novel nanopore assay to detect single cyclic oligoadenylate (cOA) molecules. This method, using a convolutional neural network, can determine cOA stoichiometry, aiding prokaryotic immune response studies.
Area of Science:
- Molecular Biology
- Biophysics
- Systems Biology
Background:
- Cyclic oligoadenylates (cOAs) are crucial second messengers in prokaryotic immunity, produced by type III CRISPR-Cas systems to control viral infections.
- cOAs activate downstream effectors, inducing dormancy or cell death to halt viral spread, with varying stoichiometries observed across different systems.
- Previous characterization of cOAs was limited to bulk measurements requiring sophisticated equipment, lacking single-molecule resolution and portable assay options.
Purpose of the Study:
- To develop a label-free, single-molecule resolution assay for detecting and characterizing cyclic oligoadenylates (cOAs).
- To determine the stoichiometric composition of cOAs produced by specific CRISPR-Cas variants.
- To explore the potential of this assay for broader applications in signaling molecule detection.
Main Methods:
- Utilized a protein nanopore assay for label-free, single-molecule detection of cOAs.
- Employed a convolutional neural network (CNN) for sensitive identification of cOA stoichiometry and mixtures.
- Validated the assay using synthetic and enzymatically produced cOA samples, including those from *Thermus thermophilus* CRISPR variants.
Main Results:
- Successfully demonstrated label-free, single-molecule detection and stoichiometric determination of cOAs.
- Identified nearly identical cOA compositions produced by CRISPR type III-A and III-B variants of *Thermus thermophilus*, confirmed by LC-MS.
- The nanopore-CNN workflow achieved single cOA resolution, outperforming previous bulk characterization methods.
Conclusions:
- The developed nanopore-CNN workflow provides a sensitive, single-molecule method for analyzing cOA stoichiometry.
- This assay overcomes limitations of bulk measurements and offers potential for portable, point-of-care applications.
- The findings offer insights into the regulation of prokaryotic immunity and can be adapted for diverse signaling molecule analyses.
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