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 fromHomo 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.

中文翻译：

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ADAR1 的 Zα 结构域以低微摩尔亲和力与 A 型核酸双链体结合

当与多种病毒和先天免疫应答蛋白中发现的 Zα 结构域结合时，DNA 和 RNA 都可以采用核酸的左手 Z 构象。虽然某些序列更喜欢采用 Z 形式，例如通常研究的 (CpG) _n重复序列，但据报道 Zα 可以与广泛的序列环境结合。研究 Zα 在 B-/A-型螺旋转化为 Z-构象之前如何与它们相互作用具有挑战性，因为结合与 Z-型采用同时发生。在这里，我们研究了智人ADAR1 的 Zα 与 (CpG) ₃构建体 (LNA (CpG) ₃ )的锁定“A 型”版本(LNA (CpG) 3 ) 的结合，其中糖皱褶被锁定到 C3'- endo /C2'中-外切构象，防止双链体采用Z 构象中发现的交替 C2'/C3'-内切糖褶皱。使用 NMR 和其他生物物理技术，我们发现 Zα ^ADAR1使用与 Z 型结合类似的界面与 LNA (CpG) _{3结合，解离常数 (} K _D ) 约为 4 μM。与 Z-DNA/Z-RNA 中每 6 bp 片段结合两个 Zα ^ADAR1相比，我们的数据表明 Zα ^ADAR1与多个 LNA 分子结合，表明完全不同的结合模式。由于 Zα ^ADAR1与非 Z 型模型的结合相对紧密，因此在进行尝试识别 Zα 结构域的 Z 型靶标的实验时，可能需要考虑其与 B/A 型螺旋的结合。在需要 Z 形采用的阴性对照的实验中，使用 LNA 结构可能是有益的。