A reprogrammed genetic code consisting of 32 distinct amino acids

Area of Science:

• Synthetic biology
• Genetic code expansion
• Protein engineering

Background:

• Sense codon reassignment allows nonproteinogenic amino acids (npAAs) incorporation.
• Current methods often limit the total number of amino acid building blocks to 20.
• Previous artificial codon box division showed promise but faced efficiency limitations.

Purpose of the Study:

• To overcome limitations in previous codon box division methods.
• To expand the genetic code beyond the standard 20 proteinogenic amino acids (pAAs).
• To facilitate multiple npAA incorporations for diverse peptide library generation.

Main Methods:

• Applied engineered tRNAs (tRNAPro1E2 and tRNAiniP) to the codon box division framework.
• Optimized translation conditions to enhance npAA incorporation efficiency.
• Utilized in vitro transcribed transfer RNAs (tRNAs) lacking nucleotide modifications.

Main Results:

• Successfully expanded the genetic code to 32 amino acids, including 11 elongator npAAs and 1 initiator npAA.
• Maintained the incorporation of all 20 standard pAAs.
• Incorporated therapeutically significant npAAs like β-amino, d-amino, and N-methyl amino acids, plus an initiator for peptide macrocyclization.

Conclusions:

• The developed platform enables the expansion of the genetic code for diverse npAA incorporation.
• This advancement facilitates the creation of novel macrocyclic peptide libraries for drug discovery.
• The platform holds significant potential for generating unique chemical entities in therapeutic development.