Recently, the research group led by Professor Shixian Lin from Life Sciences Institute, in collaboration with other teams, published a research article in ACS Central Science entitled Computationally Assisted Noncanonical Amino Acid Incorporation.
Genetic code expansion strategy utilizes orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs to recognize noncanonical amino acids (ncAAs) with unique physical, chemical, or biological functions and site-specifically install them onto the protein of interest by recoding stop codons. In order to incorporate a desired function into a protein, multiple ncAAs with such functionality had to be designed, chemically synthesized, and screened for active aaRS variants through multiple rounds of positive and negative selections. It typically takes months or even years for specialized laboratories to incorporate a new functional ncAA. In addition, the success rate of screening for active aaRS variants is also low. Therefore, there is a pressing need to develop a method that can virtually assess the recognition potential of designed ncAAs prior to initiating chemical synthesis and aaRS selection.
Figure 1. Computationally assisted noncanonical amino acid incorporation
Herein, the authors firstly systematically summarized all previously reported recognizable and unrecognizable ncAAs for four orthogonal translation systems, including PylRS/tRNA, chPheRS/tRNA, EcLeu/tRNA, and EcTyr/tRNA, respectively. Three parameters were proposed to quantify the recognition of ncAAs by aaRS: 1) hydrophobicity of ncAAs: mainly affects the efficiency of ncAAs cellular uptake; 2) solubility of ncAAs: affects the intracellular concentration of ncAAs; and 3) binding affinity of ncAAs by aaRS. Based on these parameters, the authors developed the virtual screeners for each of four orthogonal translation systems. Next, methyl ester derivatives of malonylated lysine (MalK) and glutamylated lysine (GluK) were designed and evaluated for their recognition potential by the screener, and further experiments successfully confirmed their recognition in E. coli and mammalian cells. Finally, the authors designed several Phe derivatives with strong electron-rich dialkylamino substitution groups, which were evaluated using the chPheRS/tRNA screener. These newly designed ncAAs exhibited stronger cation- π binding energy than Tyr and Phe, leading to the successful development of histone methylation Super-Readers.
The first authors of the article include Chengzhu Fang, Dr. Wenyuan Xu, Dr. Chao Liu. Professor Shixian Lin and Professor Wenlong Ding from the Fourth Affiliated Hospital of Zhejiang University School of Medicine are the corresponding authors of this article.
