In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

Henry A Lester,Dennis A Dougherty,Erik A Rodriguez

doi:10.1073/pnas.0510817103

Abstract

Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more "orthogonal" to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.

Highlights

Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure–function relationships, incorporating biophysical probes, and elucidating protein–protein interactions
To determine whether frameshift suppression is viable in Xenopus oocytes, we chose to use a tRNA that can be aminoacylated in vivo
The CUA anticodon of human serine AS (HSAS) was replaced with CCCG and ACCC to create the human serine frameshift suppressor (FS) (HSFSCCCG and HSFSACCC), which recognize the quadruplet codons CGGG and GGGU

Summary

Introduction

Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure–function relationships, incorporating biophysical probes, and elucidating protein–protein interactions. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. A remarkable variant of this approach is the use of quadruplet codons, a process that is termed frameshift suppression and was pioneered by Sisido and coworkers [6, 7] The success of this approach opens up the possibility of developing multiple additional codons, incorporating several different UAAs into a protein. To illustrate the potential of this methodology, we have successfully incorporated two and three different UAAs simultaneously into a neuroreceptor expressed in a Xenopus oocyte

Methods

Results

Conclusion