In VitroSelection Combined with Ribosomal Translation Containing Non-proteinogenic Amino Acids

Abstract

The potential applications of non-proteinogenic amino acids have increased continuously since the introduction of these molecules into a ribosomal translation system. An increasing number of studies concerning topics, such as the addition of an artificial function to a protein, cellular expression of a protein with an artificial residue, and development of an artificial peptide with a novel function, have been done using these molecules. Here, we describe recent studies that elucidate the compatibility of non-proteinogenic amino acids with ribosomal translation. We also describe the development of a simple and high-speed selection method and its potential application for the creation of a novel functional peptide with non-proteinogenic amino acids. As these studies have expanded the diversity of the artificial peptide library and increased the speed of novel functional peptide selection, they will significantly facilitate the development of new molecules, such as pharmaceutical drug candidates and bioassay probes.

Ribosomal Synthesis of Peptides with Multiple β-Amino Acids

“…Additionally, β-amino acids receive attention as the building blocks of peptide analogues for pharmaceutical uses, because β-amino acids improve the stability of the peptide analogues containing them against proteolysis. , Our study greatly increased the knowledge for the use of β-amino acids in a ribosomal translation system, so it makes β-amino acids available for the construction of the peptide library. Such highly diverse peptide libraries containing β-amino acids would become a useful source for therapeutic peptidomimetics in the future. , …”

“…It has been found that the ribosome has a remarkable tolerance for various nonproteinogenic amino acids, although natural translation system commonly employs 20 proteinogenic α-amino acids as substrates. The ribosomal tolerance toward backbone-modified amino acids, such as N -methyl amino acids and d -amino acids, has been studied, − but the results of these studies were partially inconsistent presumably because of the insufficient optimization of the cell-free translation systems to study the tolerance of ribosome. In the previous report, we successfully applied our optimized translation system to reveal d -amino acids compatibility with ribosomal translation, and found various d -amino acids that could be used for the ribosomal peptide synthesis.…”

“…Our study elucidated β-amino acid compatibility with ribosomal translation, which would make it possible to create nonstandard peptide libraries containing β-amino acids. In the future, the combination of a ribosomal translation system with β-amino acids and an in vitro selection method, , such as our high-speed selection method (TRAP display, transcription-translation coupled with the association of a puromycin linker), will allow the rapid development of therapeutic peptidomimetics from these highly diverse nonstandard peptide libraries. , …”

Genetic Code Expansion: A Brief History and Perspective

“…The existence of mutant E. coli ribosomes that enhance d -amino acid incorporation in vitro indicates potential for in vivo incorporation of d -amino acids . Similarly, in 2018, the Murakami lab improved incorporation of multiple β-amino acids, producing peptides with natural amino acid spacers between two or three β-amino acids in their optimized translation system . Translating the in vitro advances in incorporation of backbone analogue UAAs to cells should catch on as more suitable aaRS/tRNA pairs are established.…”

“…67 Similarly, in 2018, the Murakami lab improved incorporation of multiple β-amino acids, producing peptides with natural amino acid spacers between two or three β-amino acids in their optimized translation system. 68 Translating the in vitro advances in incorporation of backbone analogue UAAs to cells should catch on as more suitable aaRS/ tRNA pairs are established. In 2019, Dedkova and Hecht found that wild-type PylRS could incorporate a fluorescent oxazole UAA lacking an asymmetric center or α-amino group.…”