The present invention is in the field of a compound for use as a medicament for treatment of tRNA deficiencies in living cells, a dosage comprising said compound, and an in vivo and in vitro method for treatment of tRNA deficiencies, as well as for prevention, mitigation of symptoms, and regeneration of cells.

The disclosure provides methods of making a protein having a desired non-standard amino acid incorporated at its N-terminus in a cell and methods of screening for an amino acyl tRNA synthetase variant that preferentially selects a non-standard amino acid against its standard amino acid counterpart or undesired non-standard amino acids for incorporation into a protein in a cell.

Compositions, systems, and methods for preparation of polypeptides having multiple iterations of non-standard amino acids are provided. The compositions and method can be used to produce recombinant proteins at a greater yield than the same or similar polypeptides made using conventional compositions, systems, and methods. Accordingly, in some embodiments, the polypeptides are ones that could not be made using conventional methods and reagents, or could not be made a sufficient yield or purity to serve a practical purpose using conventional methods and reagents. Polypeptides made using the disclosed compositions, systems, and methods are also provided.

Provided are compositions comprising newly identified protein fragments of aminoacyl-tRNA synthetases, polynucleotides that encode them and complements thereof, related agents, and methods of use thereof in diagnostic, drug discovery, research, and therapeutic applications.

The disclosure provides amino acid sequence variants of orthogonal aminoacyl-tRNA synthetases (AARSs) having increased activity and selectivity compared to previous AARSs, and methods of producing the same.

Mutant aminoacyl-tRNA synthetase (aaRS) proteins are provided. Nucleic acid molecules encoding the mutant aaRSs, orthogonal translation systems comprising the mutant aaRSs or nucleic acid molecules, cells comprising the orthogonal translation systems, as well as methods of using same are also provided.

The present invention relates to an isolated nucleotide acid sequence comprising a) the cDNAs of two strands of a target double-stranded RNA (dsRNA) separated by an autocatalytic intron flanked by exon fragments, and b) a plant viroid, wherein element (a) is inserted in the plant viroid sequence. The expression of this nucleotide sequence in a host cell, such as E. coli, along with a tRNA ligase, allows the production of high amounts of dsRNA specific for a target gene. This dsRNA can be used in the interfering RNA technology for silencing gene expression. Additionally, the present invention also relates to a method for producing said dsRNA.

The present invention describes methods to create chimeric aminoacyl-tRNA synthetases (aaRS) derived from bacteria which show optimal activity and high thermostability. These chimeric aaRSs can be more aggressively engineered to generate a wider assortment of Uaa-selective mutants that are stable at the physiological temperature. The invention further describes the composition of chimeric TyrRSs, generated from E. coli and G. stearothermophilus TyrRSs, which demonstrate enhanced stability relative to EcTyrRS and higher activity relative to both TyrRS enzymes.

The disclosure provides amino acid sequence variants of orthogonal aminoacyl-tRNA synthetases (AARSs) having increased activity and selectivity compared to previous AARSs, and methods of producing the same.

Another aim of the current invention may include a recombinant cell-free expression system, the reaction mixture containing all the cell-free reaction components necessary for the in vitro biosynthesis of biological compounds, proteins, enzymes, biosimilars or chemical modification of small molecules.