title: A semi-synthetic organism with an expanded genetic alphabet.
abstract: Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Denis A
authors.initials: DA
authors.lastname: Malyshev
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Kirandeep
authors.initials: K
authors.lastname: Dhami
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lavergne
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Tingjian
authors.initials: T
authors.lastname: Chen
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Foster
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Floyd E
authors.initials: FE
authors.lastname: Romesberg
mesh_terms: D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005815:Genetic Code / Q000187:drug effects / Q000235:genetics*; D042822:Genomic Instability / Q000235:genetics*; D007546:Isoquinolines / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D033704:Nucleotide Transport Proteins / Q000235:genetics / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*; D013871:Thiones / Q000378:metabolism
chemical_list: D020418:Algal Proteins; D003470:Culture Media; D007546:Isoquinolines; D009281:Naphthalenes; D033704:Nucleotide Transport Proteins; D009711:Nucleotides; D013871:Thiones