Nanopores Reveal the Stoichiometry of Single Oligoadenylates Produced by Type III CRISPR-Cas

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 singlemolecule 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.

Understanding Electrophoresis and Electroosmosis in Nanopore Sensing with the Help of the Nanopore Electro-Osmotic Trap

“…Nanopore technology has been successfully applied to study various biomolecules at the single-molecule level, e.g., DNA/RNA sequencing, , protein fingerprinting, , peptide sequencing, , and metabolite detection. , The major kinetic processes, i.e., the capture, residence, and escape of analytes, universally exist in these nanopore sensing technologies. For example, in simple translocation experiments, target molecules are captured by the electric field whereupon they translocate through the nanopore; in some experiments with biological nanopores, proteins are captured and trapped by electro-osmotic flow (EOF); , for DNA or peptide sequencing, a DNA helicase is docked onto a biological pore; , in small solid-state nanopores, proteins are captured and escape again; for the detection of small chemicals, DNA-tethered streptavidin is docked on a nanopore; ,, for the trapping of proteins, a DNA-origami structure is docked on solid-state nanopores, − et cetera.…”