Halogen Bonding as a Molecular Recognition Strategy for Genetic Code Expansion

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Aminoacyl-tRNA synthetases (aaRSs) are crucial for protein synthesis, attaching specific amino acids (AAs) to tRNAs.
• Genetic code expansion (GCE) allows incorporating non-canonical amino acids (ncAAs) into proteins, creating novel structures and functions.
• Recognition of ncAAs by aaRSs is key for GCE, but challenging due to structural similarities.

Purpose of the Study:

• To investigate if halogen bonding (XB) can mediate the recognition of halogenated ncAAs by aaRSs.
• To engineer aaRSs for the selective incorporation of 3,5-diiodo-l-tyrosine (DITY) via GCE.
• To demonstrate that aaRSs can discriminate between structurally similar halogenated ncAAs.

Main Methods:

• Protein engineering of aaRSs to recognize halogenated ncAAs.
• In vitro assays to assess aaRS specificity for DITY and related compounds.
• In vivo translation systems to confirm selective ncAA incorporation during protein synthesis.

Main Results:

• Engineered aaRSs selectively recognized 3,5-diiodo-l-tyrosine (DITY).
• The engineered aaRSs demonstrated discrimination against 3,5-dichloro-, 3,5-dibromo-, and 3,5-dimethyl-l-tyrosine.
• Halogen bonding (XB) was confirmed as the mechanism for ncAA recognition by aaRSs.
• Successful incorporation of DITY into proteins via GCE was achieved.

Conclusions:

• Halogen bonding (XB) is a viable mechanism for expanding the genetic code with ncAAs.
• Engineered aaRSs can achieve high selectivity for structurally similar ncAAs based on halogenation.
• This work provides a foundation for designing novel proteins with tailored functions through GCE.