Transcription activator-like effector (TALE) proteins have been used extensively for targeted binding of fusion proteins to loci of interest in (epi)genome engineering. Such approaches typically utilize four canonical TALE repeat variable diresidue (RVD) types, corresponding to the identities of two key amino acids, to target each nucleotide. Alternate RVDs with improved specificity are desired. Here, we focused on seven noncanonical RVDs that have been suggested to have improved specificity for their target nucleotides. We used custom protein binding microarrays to characterize the DNA-binding activity of 65 TALEs containing these alternate or corresponding canonical RVDs at multiple positions to ~5,000 unique DNA sequences per protein. We found that none of the noncanonical thymine-targeting RVDs displayed stronger preference for thymine than did the canonical RVD. Of the noncanonical RVDs with putatively improved specificity for guanine, only EN and NH showed greater discrimination of guanine over adenine. This improved specificity, however, comes at a cost: more substitutions of a noncanonical RVD for a canonical RVD generally decreased the protein's DNA-binding activity. Our results highlight the need to investigate RVD-nucleotide specificities in multiple protein contexts and suggest that a balance between canonical and noncanonical RVDs is needed to build TALEs with improved specificity.

中文翻译：

---

上下文和非规范重复可变残基的数量阻碍了具有改进的DNA靶向性的TALE蛋白的设计。

转录激活因子样效应物（TALE）蛋白已广泛用于融合蛋白与（epi）基因组工程中目标基因座的靶向结合。此类方法通常利用四种典型的TALE重复可变双残基（RVD）类型（对应于两个关键氨基酸的身份）来靶向每个核苷酸。需要具有改善的特异性的替代RVD。在这里，我们集中于七个非规范的RVD，已被建议对其目标核苷酸具有更高的特异性。我们使用定制的蛋白质结合微阵列来表征65个TALE的DNA结合活性，这些TALE在多个位置包含这些交替或对应的规范RVD，每个蛋白质约有5,000个独特的DNA序列。我们发现，非经典的针对胸腺嘧啶的RVD都没有比经典的RVD表现出更强的对胸腺嘧啶的偏好。在公认的鸟嘌呤特异性非经典RVD中，只有EN和NH对鸟嘌呤的区分度高于腺嘌呤。但是，这种提高的特异性需要付出一定的代价：用非典范RVD替代典范RVD通常会降低蛋白质的DNA结合活性。我们的结果强调需要在多种蛋白质环境中研究RVD核苷酸的特异性，并建议需要在规范性和非规范性RVD之间建立平衡，以构建具有更高特异性的TALE。但是，这种提高的特异性需要付出一定的代价：用非典型RVD替代典型RVD通常会降低蛋白质的DNA结合活性。我们的结果强调需要在多种蛋白质环境中研究RVD核苷酸的特异性，并建议需要在规范性和非规范性RVD之间建立平衡，以构建具有更高特异性的TALE。但是，这种提高的特异性需要付出一定的代价：用非典型RVD替代典型RVD通常会降低蛋白质的DNA结合活性。我们的结果强调需要在多种蛋白质环境中研究RVD核苷酸的特异性，并建议需要在规范性和非规范性RVD之间建立平衡，以构建具有更高特异性的TALE。