Disclosed are engineered polynucleotides, engineered ribosomes comprising the engineered polynucleotides, engineered cells and systems comprising the engineered polynucleotides and ribosomes, and methods of making and using the engineered polynucleotides, engineered ribosomes, engineered cells and systems. The engineered polynucleotides, engineered ribosomes, and engineered cells may be utilized to prepare sequence defined polymers and to select for mutant ribosomes that are capable of incorporating non-canonical amino acids into a polymer.

Engineered 23S rRNAs and methods of use thereof for translation of proteins incorporating non-standard amino acids are provided. Typically, the 23S rRNA includes one or more mutations at positions 2496-2507 relative to E. coli wildtype 23S rRNA, wherein a ribosome composed of the 23S rRNA can catalyze the covalent transfer of a non-standard amino acid from an aminoacyl-RNA onto a nascent peptide chain. For example, the 23S rRNA can include the sequence UGACUU at positions 2502-2507 relative to E. coli wildtype 23S rRNA, and optionally the sequence AGCGUGA from positions 2057-2063 relative to E. coli wildtype 23S rRNA. The 23S rRNA can include additional or alternative deletions, substitutions, insertions, or combination thereof. The compositions and methods can be used to make polypeptides and sequence defined polymers.

Disclosed are engineered or modified E. coli ribosomes and methods, components, compositions, and kits for preparing and identifying engineered or modified E. coli ribosomes. The engineered or modified E. coli ribosomes may be prepared and identified under a set of defined conditions, such as in the presences of a engineered or modified tRNA comprising a non-natural, non-α-amino acid monomer (NNA), in order to obtain an engineered or modified ribosome that utilizes the engineered or modified tRNA as a substrate for synthesizing a polymer comprising the NNA.

Compositions and methods relating to bacterial ribosomes selected to increase the incorporation of at least one glycosylated amino acid into a protein versus a wild-type bacterial ribosome. Selection embodiments include growing bacteria in the presence of a puromycin derivative, wherein a surviving clone has a ribosome that incorporates at least one glycosylated amino acid into a protein.

Embodiments of the present disclosure pertain to methods of synthesizing a peptide by associating at least one isolated ribosomal RNA (rRNA) fragment with a plurality of transfer RNA fragments linked to amino acids (tRNA fragments). The at least one rRNA fragment includes at least one domain that associates with the tRNA fragments. The at least one rRNA fragment catalyzes the synthesis of the peptide through peptidyl transfer of the amino acids from the tRNA fragments independently of messenger RNA (mRNA) templates. Additionally, the peptidyl transfer occurs independently of proteins. Further embodiments of the present disclosure pertain to systems for synthesizing a peptide. Such systems include the rRNA fragments of the present disclosure and optionally a plurality of tRNA fragments.

Engineered 23S rRNAs and methods of use thereof for translation of proteins incorporating non-standard amino acids are provided. Typically, the 23S rRNA includes one or more mutations at positions 2496-2507 relative to E. coli wildtype 23S rRNA, wherein a ribosome composed of the 23S rRNA can catalyze the covalent transfer of a non-standard amino acid from an aminoacyl-RNA onto a nascent peptide chain. For example, the 23S rRNA can include the sequence UGACUU at positions 2502-2507 relative to E. coli wildtype 23S rRNA, and optionally the sequence AGCGUGA from positions 2057-2063 relative to E. coli wildtype 23S rRNA. The 23S rRNA can include additional or alternative deletions, substitutions, insertions, or combination thereof. The compositions and methods can be used to make polypeptides and sequence defined polymers.

Disclosed are engineered or modified E. coli ribosomes and methods, components, compositions, and kits for preparing and identifying engineered or modified E. coli ribosomes. The engineered or modified E. coli ribosomes may be prepared and identified under a set of defined conditions, such as in the presences of a engineered or modified tRNA comprising a non-natural, non--amino acid monomer (NNA), in order to obtain an engineered or modified ribosome that utilizes the engineered or modified tRNA as a substrate for synthesizing a polymer comprising the NNA.