Abstract: Disclosed herein are methods, cells, engineered microorganisms, and kits for increased production of a nucleic acid molecule that comprises an unnatural nucleotide.

Abstract: Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.

Abstract: Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

Abstract: Disclosed herein are compositions, methods, cells, engineered microorganisms, and kits for increasing the production of proteins or polypeptides comprising one or more unnatural amino acids. Further provided are compositions, cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.

Abstract: Disclosed herein are methods of reverse transcribing a polynucleotide comprising an unnatural ribonucleotide comprising reverse transcribing the polynucleotide with a reverse transcriptase in the presence of an unnatural dNTP comprising an unnatural nucleobase, wherein the reverse transcriptase polymerizes cDNA into which the unnatural NTP is incorporated. In some embodiments, the polynucleotide is present at a concentration less than or equal to about 500 nM and/or the polynucleotide is a tRNA, mRNA, RNA aptamer, or a member of a plurality of RNA aptamer candidates.

Abstract: Disclosed herein are proteins, methods, cells, engineered microorganisms, and kits for generating a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum. Also disclosed herein proteins, methods, cells, engineered microorganisms, and kits for production of a nucleic acid molecule that comprises an unnatural nucleotide utilizing a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum.

Abstract: Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

Abstract: Disclosed herein are methods, cells, engineered microorganisms, and kits for increased production of a nucleic acid molecule that comprises an unnatural nucleotide.

Abstract: A recombinantly expressed nucleotide triphosphate transporter efficiently imports the triphosphates of unnatural nucleotides into cells, and the endogenous cellular machinery incorporates those nucleotides into cellular nucleic acids. UBPs can therefore form within the cell's nucleic acids. Moreover, neither the presence of the unnatural triphosphates nor the replication of the UBP represents a significant growth burden. The UBP is not efficiently excised by nucleic acid repair pathways, and therefore can be retained as long as the unnatural triphosphates are available in the growth medium. Thus, the resulting cell is the first organism to stably propagate an expanded genetic alphabet.

Abstract: Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.

Abstract: A recombinantly expressed nucleotide triphosphate transporter efficiently imports the triphosphates of unnatural nucleotides into cells, and the endogenous cellular machinery incorporates those nucleotides into cellular nucleic acids. UBPs can therefore form within the cell's nucleic acids. Moreover, neither the presence of the unnatural triphosphates nor the replication of the UBP represents a significant growth burden. The UBP is not efficiently excised by nucleic acid repair pathways, and therefore can be retained as long as the unnatural triphosphates are available in the growth medium. Thus, the resulting cell is the first organism to stably propagate an expanded genetic alphabet.

Abstract: Disclosed herein are compositions, methods, and kits for a cell incorporating unnatural amino acids into an unnatural polypeptide. Also disclosed herein are compositions, methods, and kits for increasing activity and yield of the unnatural polypeptide synthesized by the cell.

Abstract: Provided herein are eukaryotic semi-synthetic organisms and their methods of use and manufacture.

Abstract: Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

Abstract: Disclosed herein are compositions, methods, cells, engineered microorganisms, and kits for increasing the production of proteins or polypeptides comprising one or more unnatural amino acids. Further provided are compositions, cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.

Abstract: Disclosed herein are methods, cells, engineered microorganisms, and kits for increased production of a nucleic acid molecule that comprises an unnatural nucleotide.

Abstract: Disclosed herein are methods, cells, engineered microorganisms, and kits for increasing the production of polypeptides comprising one or more unnatural amino acids. Further provided are cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.

Abstract: Disclosed herein are proteins, methods, cells, engineered microorganisms, and kits for generating a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum. Also disclosed herein proteins, methods, cells, engineered microorganisms, and kits for production of a nucleic acid molecule that comprises an unnatural nucleotide utilizing a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum.

Abstract: Disclosed herein are in vivo methods, compositions, and kits for producing nucleic acids which comprises at least one unnatural base. Disclosed herein are in vivo methods of producing a nucleic acid with an expanded genetic alphabet, the method comprising incorporatin at least one unnatural base in the nucleic acid. Disclosed herein are semi-synthetic organisms comprising an expanded genetic alphabet, wherein the genetic alphabet comprises at least one unnatural base. Disclosed herein are compositions that include a heterologous or recombinant polymerase and methods of use thereof. Further, disclosed herein are kits that are useful for stably incorporating an unnatural nucleic acid into a nucleic acid molecule, e.g., using the methods provided by the present invention in in vitro condition or under a cell free condition.

Abstract: Disclosed herein are proteins, methods, cells, engineered microorganisms, and kits for generating a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum. Also disclosed herein proteins, methods, cells, engineered microorganisms, and kits for production of a nucleic acid molecule that comprises an unnatural nucleotide utilizing a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum.