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.

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The transition to the third dimension enables the creation of spintronic nanodevices with significantly enhanced functionality compared to traditional 2D magnetic applications. In this study, we extend common two-dimensional magnetic vortex configurations, which are known for their efficient dynamical response to external stimuli without a bias magnetic field, into the third dimension. This extension results in a substantial increase in vortex frequency, reaching up to 5 GHz, compared to the typical sub-GHz range observed in planar vortex oscillators. A systematic study reveals a complex pattern of vortex excitation modes, explaining the decrease in the lowest gyrotropic mode frequency, the inversion of vortex mode intensities, and the nontrivial spatial distribution of vortex dynamical magnetization noted in previous research. These phenomena enable the optimization of both oscillation frequency and frequency reproducibility, minimizing the impact of uncontrolled size variations in those magnetic nanodevices.@en