Zα Domain of ADAR1 Binds to an A-Form-like Nucleic Acid Duplex with Low Micromolar Affinity.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG) n repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption. Here, we studied the binding of Zα from Homo sapiens ADAR1 to a locked "A-type" version of the (CpG) 3 construct (LNA (CpG) 3 ) where the sugar pucker is locked into the C3'- endo /C2'- exo conformation, which prevents the duplex from adopting the alternating C2'/C3'- endo sugar puckers found in the Z-conformation. Using NMR and other biophysical techniques, we find that Zα ADAR1 binds to the LNA (CpG) 3 using a similar interface as for Z-form binding, with a dissociation constant ( K D ) of ∼4 μM. In contrast to Z-DNA/Z-RNA, where two Zα ADAR1 bind to every 6 bp stretch, our data suggests that Zα ADAR1 binds to multiple LNA molecules, indicating a completely different binding mode. Because Zα ADAR1 binds relatively tightly to a non-Z-form model, its binding to B/A-form helices may need to be considered when experiments are carried out which attempt to identify the Z-form targets of Zα domains. The use of LNA constructs may be beneficial in experiments where negative controls for Z-form adoption are needed.