Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies,ACS Synthetic Biology

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Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-01-21 , DOI: 10.1021/acssynbio.1c00471
Eike F Joest ₁ , Christian Winter ₁ , Joshua S Wesalo ₂ , Alexander Deiters ₂ , Robert Tampé ₁

Affiliation

Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

中文翻译：

通过核糖体跳跃有效抑制琥珀原位合成光条件纳米抗体

遗传密码扩展是修饰蛋白质原位合成的通用方法。在 mRNA 翻译过程中，琥珀终止密码子被抑制以位点特异性地掺入非规范氨基酸。因此，纳米抗体可以配备光笼氨基酸来根据需要控制靶标结合。琥珀抑制和蛋白质合成的效率可能会随着不可预测的背景表达而变化，其原因很难理解。在这里，我们发现了一个重大限制，阻止了具有用于光控制的 N 端修饰的纳米抗体的合成。经过系统分析，我们假设纳米抗体的合成在翻译的早期阶段受到核糖体不准确的严重影响。为了避免过早终止密码子的背景导致通读，我们设计了一种基于核糖体跳跃的新抑制概念。例如，我们在哺乳动物细胞中生成了具有光激活靶标结合的胞内抗体。这些发现为遗传密码扩展提供了宝贵的见解，并描述了生成修饰纳米抗体的通用合成路线，为化学工具的高效位点特异性整合开辟了新的视角。在光药理学领域，我们灵活的体内概念构建了一个调节靶蛋白功能和相互作用的理想平台。

更新日期：2022-01-21

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