Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes

Abstract

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

Cell-sized mechanosensitive and biosensing compartment programmed with DNA

“…Modern TXTL platforms are used for medicine and biomolecular manufacturing, such as the production of vaccine and therapeutics 5, 6 . By performing non-natural chemistries 3, 7, 8 , the TXTL technology has been improved to expand the molecular repertoire of biological systems. Because the time for design-build-test cycle is dramatically reduced, TXTL has become a powerful platform to rapidly prototype genetic programs in vitro , from testing single regulatory elements to recapitulating metabolic pathways 9 .…”

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation

“…[17] On the other hand, the topologically controlled synthesis of multicyclic peptides linked via disulfide bonds by using ribosomes has been challenging due to near-random crosslinking of Cys residues. [1f, 7] To overcome this limitation, we utilized our genetic code reprogramming approach using flexizymes integrated with ac ustom-made in vitro translation apparatus (referred to the FIT system), [9,10] and demonstrated the ribosomal synthesis of bi-and tricyclic peptides.T his approach relies on preferential thioether macrocyclization where the second Cys residue (underlined) in ClAc-XC-C-Cs electively reacts with N-terminal ClAc group to form the smallest cyclic peptide via at hioether bond. Ther emaining two Cys residues oxidatively form ad isulfide bond to yield ab icyclics caffold.…”

“…Owing to the higher nucleophilicity of selenocysteine (Sec) over cysteine (Cys), Sec can preferentially react with halogenated residues to achieve regioselective cyclization of peptides containing both Sec and Cys residues ( Figure 1). Moreover,b yt aking advantage of genetic code reprogramming using flexizymes integrated with acustomized reconstituted translation system (FIT system), [9,10] both Sec and the halogenated residues can be incorporated into peptide chains,t hus enabling the regioselective cyclization of biosynthetic peptides into the desirable bicyclic or tricyclic peptide scaffolds described above.This work will benefit the development of ribosomally expressed topologically controlled multicyclic peptide libraries. Moreover,b yt aking advantage of genetic code reprogramming using flexizymes integrated with acustomized reconstituted translation system (FIT system), [9,10] both Sec and the halogenated residues can be incorporated into peptide chains,t hus enabling the regioselective cyclization of biosynthetic peptides into the desirable bicyclic or tricyclic peptide scaffolds described above.This work will benefit the development of ribosomally expressed topologically controlled multicyclic peptide libraries.…”

“…By varying the pattern of the crosslinking residues (i.e., Sec,C ys,a nd halogenated residues) and the secondary crosslinks (i.e., alkylation or arylation reagents), multicyclic peptide scaffolds can be designed and synthesized in ac ontrolled manner ( Figure 1). Moreover,b yt aking advantage of genetic code reprogramming using flexizymes integrated with acustomized reconstituted translation system (FIT system), [9,10] both Sec and the halogenated residues can be incorporated into peptide chains,t hus enabling the regioselective cyclization of biosynthetic peptides into the desirable bicyclic or tricyclic peptide scaffolds described above.This work will benefit the development of ribosomally expressed topologically controlled multicyclic peptide libraries.…”

Nucleic Acid‐Barcoding Technologies: Converting DNA Sequencing into a Broad‐Spectrum Molecular Counter

“…[77] This eliminates competition between natural and unnatural codon assignments and permits multiple unique UAAs to be installed in asingle peptide. [78] However, am ajor limitation associated with genetic-code reprogramming that mRNAd isplay cannot overcome is the need to generate UAA-charged tRNAs.T his is typically accomplished enzymatically by utilizing directed evolution to create aminoacyl-tRNAsynthetases for aU AA of interest [79] or chemically by either ligating modified amino-acid buildingblocks to tRNAm olecules [80] or modifying the amino acid moiety of natural aminoacylated tRNAs. [81] While enzymatic tRNAc harging can be performed during the IVT reaction, the chemical approach requires pre-synthesized aminoacylated tRNAs to be added to the IVT reaction for ribosomal incorporation into peptides.Notably,these strategies require unique chemical approaches or evolved tRNAsynthetases for each UAAofinterest.…”