About Cyclic tetra-AMP

Cyclic tetra-AMP, also known as cyclic tetra-adenylate or cA₄, is a second messenger found in bacteria and archaea that is involved in antiviral immunity mediated by type III CRISPR-Cas systems.^1-3

Synthesis

Cyclic oligoadenylates (cOAs), including cyclic tetra-AMP and cyclic hexa-AMP, are synthesized from ATP by the Cas10 subunit of the type III effector complex upon effector complex recognition of target bacteriophage or plasmid RNA transcripts (Figure 1).^1-3 Eventual cleavage of the target RNA by an RNase within the effector complex causes Cas10 to return to its inactive state, turning off cOA synthesis.

Figure 1. In type III CRISPR-Cas systems, type III effector complexes comprised of Cas proteins and a CRISPR RNA (crRNA) guide detect target RNA, inducing production of cyclic tetra-AMP and other cyclic oligoadenylates from ATP by the Cas10 subunit. Cyclic tetra-AMP binds to and activates effectors, including Csm6 and Csx1 ribonucleases, to provide antiviral immunity and induce host dormancy. Host ring nucleases then degrade cyclic tetra-AMP into linear oligoadenylate species to turn off the antiviral response and allow exit from dormancy.

Function

cOAs act as second messengers to mount an antiviral response by binding to and activating effectors possessing CRISPR-associated Rossmann-fold (CARF) domains, most notably the RNases Csm6 (in type III-A systems) and Csx1 (in type III-B systems).^1-4 Activated Csm6 and Csx1 enzymes degrade both invader and host cell RNA to prevent replication of the invader and induce dormancy of the host cell (Figure 1). The cOA responsible for activating Csm6 and Csx1 varies by species.⁵ For example, cyclic tetra-AMP activates Csm6 from Thermus thermophilus and Csx1 from the archaeon Sulfolobus solfataricus, whereas cyclic hexa-AMP activates Csm6 from Enterococcus italicus and Streptococcus thermophilus.

Cyclic tetra-AMP can activate numerous additional CARF domain-containing nucleases such as the metal-dependent DNA nuclease Can1, which nicks supercoiled DNA, Can2, which has DNA nickase and RNase activities, and Card1, which cleaves single-stranded DNA and RNA.^4-6 Several of these nucleases have been shown to provide antiviral immunity and/or induce growth arrest in infected host cells. Some effectors activated by cyclic tetra-AMP have non-nuclease activities, including membrane proteins and the CalpL protease, which notably lacks a CARF domain.^7-9

Degradation

Cyclic tetra-AMP is eventually degraded to linear oligoadenylate species by ring nucleases to turn off the antiviral response (Figure 1).^2,3,10 As mentioned above, Csm6 is a CARF domain-containing effector that is activated by cyclic tetra-AMP in specific species, however it also acts as a self-limiting enzyme and ring nuclease by degrading cyclic tetra-AMP.^3,11 In S. solfataricus, the Crn1 family of ring nucleases degrades cyclic tetra-AMP, causing deactivation of the cyclic tetra-AMP-dependent effector Csx1.^2,12

Certain viruses also encode ring nucleases as anti-CRISPR countermeasures to neutralize the host cell's antiviral defenses.¹² AcrIII-1 is a ring nuclease found in numerous archaeal and bacterial viruses that degrades cyclic tetra-AMP and can neutralize cyclic tetra-AMP-dependent type III CRISPR antiviral defense systems.

Measuring Cyclic tetra-AMP

Cayman's new Cyclic tetra-AMP ELISA Kit provides a straightforward, reliable way to quantify cyclic tetra-AMP levels in bacterial lysates. Whether you're working to identify cyclic tetra-AMP-producing bacterial species, study the induction, regulation, and suppression of type III CRISPR-Cas systems, or identify and characterize novel host or viral ring nucleases, this new kit can help you advance understanding of type III CRISPR-Cas systems and antiviral immunity in bacteria and archaea, as well as viral countermeasures against these systems.

About the Assay

Cayman's Cyclic tetra-AMP ELISA Kit is an easy-to-use competitive ELISA for the quantification of cyclic tetra-AMP in bacterial cell lysates. This kit includes all necessary reagents and uses a simple protocol that provides rapid results in under three hours.

The assay is based on the competition between free cyclic tetra-AMP in samples (or standards) and a cyclic tetra-AMP-HRP conjugate (cyclic tetra-AMP-HRP Tracer) for binding to a limited amount of cyclic tetra-AMP polyclonal antibody binding sites. In one single-step incubation, samples are added to IgG-coated wells along with the specific cyclic tetra-AMP polyclonal antiserum and the cyclic tetra-AMP-HRP Tracer (Figure 2, step 1). After two hours of incubation, plates are washed to remove unbound reagents and developed with the TMB Substrate Solution containing an HRP substrate (Figure 2, steps 2 and 3). This results in the formation of a colorimetric product that can be detected by a plate reader at 450 nm. The absorbance intensity is proportional to the amount of cyclic tetra-AMP-HRP Tracer bound to the well, and therefore inversely proportional to the concentration of cyclic tetra-AMP in the tested sample.

Figure 2. Schematic of the competitive ELISA. For the complete assay protocol, please refer to the kit booklet (PDF).

High Sensitivity and Specificity

Cayman's Cyclic tetra-AMP ELISA Kit uses a cyclic tetra-AMP polyclonal antiserum that offers excellent sensitivity and low cross-reactivity to structurally related molecules. The assay has a range of 0.080-500 nM, with a midpoint (50% B/B₀) of 7.168 nM, a sensitivity (80% B/B₀) of 0.853 nM, and a lower limit of detection (LLOD) of 0.112 nM (Figure 3).

Figure 3. Typical standard curve.

This kit demonstrates a high degree of specificity, with minimal detection of cyclic hexa-AMP, cyclic di-AMP, and cyclic AMP (cAMP).

Cross reactivity of the Cyclic tetra-AMP ELISA Kit

For additional information on the performance of this kit, including parallelism, spike and recovery, and linearity data, please refer to the kit booklet (PDF).

Learn more about how Cayman defines key ELISA performance metrics and validates our assays in our article A Quick Glance at Competitive ELISA Performance Characteristics & Validation.

The Cayman Advantage

At Cayman, we have an exceptional understanding of assay development, validation, and performance. Each of our assay kits undergoes rigorous quality testing to certify high precision and accuracy to deliver the sensitivity and specificity needed to detect biologically significant analyte levels. Our attention to these details ensures you will obtain reproducible results, from day to day and lot to lot, with expert technical support readily available to assist you. Learn more about the Cayman Advantage in our article, Why Cayman Assay Kits.

Our Cyclic tetra-AMP ELISA Kit is also complemented by our comprehensive CDN product line, which includes CDNs and negative controls, inhibitors and activators of cGAS-STING signaling, and additional ELISA kits to measure the CDNs 2'3'-cGAMP, 3'3'-cGAMP, 3'2'-cGAMP, cyclic di-GMP, and cyclic di-AMP. Explore the resources below to discover our full line of CDN signaling products.

You May Also Be Interested In

1. Kazlauskiene, M., Kostiuk, G., Venclovas, Č., et al. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357(6351), 605-609 (2017).
2. Athukoralage, J.S., Rouillon, C., Graham, S., et al. Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature 562(7762), 277-280 (2018).
3. Stella, G. and Marraffini, L. Type III CRISPR-Cas: Beyond the Cas10 effector complex. Trends Biochem. Sci. 49(1), 28-37 (2024).
4. Rostøl, J.T., Xie, W., Kuryavyi, V., et al. The Card1 nuclease provides defence during type III CRISPR immunity. Nature 590(7847), 624-629 (2021).
5. McMahon, S.A., Zhu, W., Graham, S., et al. Structure and mechanism of a type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate. Nat. Commun. 11(1), 500 (2020).
6. Zhu, W., McQuarrie, S., Grüschow, S., et al. The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence. Nucleic Acids Res. 49(5), 2777-2789 (2021).
7. Grüschow, S., McQuarrie, S., Ackermann, K., et al. CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23. Nucleic Acids Res. 52(6), 2761-2775 (2024).
8. Baca, C.F., Yu, Y., Rostøl, J.T., et al. The CRISPR effector Cam1 mediates membrane depolarization for phage defence. Nature 625(7996), 797-804 (2024).
9. Rouillon, C., Schneberger, N., Chi, H., et al. Antiviral signaling by a cyclic nucleotide activated CRISPR protease. Nature 614(7946), 168-174 (2023).
10. Samolygo, A., Athukoralage, J.S., Graham, S., et al. Fuse to defuse: A self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence. Nucleic Acids Res. 48(11), 6149-6156 (2020).
11. Jia, N., Jones, R., Yang, G., et al. CRISPR-Cas III-A Csm6 CARF domain is a ring nuclease triggering stepwise cA₄ cleavage with ApA>p formation terminating RNase activity. Mol. Cell 75(5), 944-956 (2019).
12. Athukoralage, J.S., McMahon, S.A., Zhang, C., et al. An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity. Nature 277(7791), 572-575 (2020).