Department of Chemistry, Virginia Commonwealth University, 1001 W Main St., Richmond, VA, 23284, USA.
Massey Cancer Center, Virginia Commonwealth University, 401 College St., Richmond, VA, 23219, USA.
Nat Commun. 2023 Aug 17;14(1):5008. doi: 10.1038/s41467-023-40529-x.
Genetic code expansion (GCE) offers many exciting opportunities for the creation of synthetic organisms and for drug discovery methods that utilize in vitro translation. One type of GCE, sense codon reassignment (SCR), focuses on breaking the degeneracy of the 61 sense codons which encode for only 20 amino acids. SCR has great potential for genetic code expansion, but extensive SCR is limited by the post-transcriptional modifications on tRNAs and wobble reading of these tRNAs by the ribosome. To better understand codon-tRNA pairing, here we develop an assay to evaluate the ability of aminoacyl-tRNAs to compete with each other for a given codon. We then show that hyperaccurate ribosome mutants demonstrate reduced wobble reading, and when paired with unmodified tRNAs lead to extensive and predictable SCR. Together, we encode seven distinct amino acids across nine codons spanning just two codon boxes, thereby demonstrating that the genetic code hosts far more re-assignable space than previously expected, opening the door to extensive genetic code engineering.
遗传密码扩展（GCE）为创建合成生物体和利用体外翻译的药物发现方法提供了许多令人兴奋的机会。一种类型的 GCE，即同义密码子重排（SCR），专注于打破编码仅 20 种氨基酸的 61 个同义密码子的简并性。SCR 在遗传密码扩展方面具有很大的潜力，但广泛的 SCR 受到 tRNA 的转录后修饰和核糖体对这些 tRNA 的摆动阅读的限制。为了更好地理解密码子-tRNA 配对，我们在这里开发了一种测定法来评估氨酰-tRNA 相互竞争特定密码子的能力。然后我们表明，超精确的核糖体突变体显示出降低的摆动阅读，并且当与未经修饰的 tRNA 配对时，会导致广泛且可预测的 SCR。总之，我们在跨越仅两个密码子盒的九个密码子中编码了七个不同的氨基酸，从而证明遗传密码具有比以前预期更多的可重新分配空间，为广泛的遗传密码工程打开了大门。
