title: Site-specific incorporation of unnatural amino acids into receptors expressed in Mammalian cells.
abstract: We describe an approach to achieve unnatural amino acid incorporation into channels and receptors expressed in mammalian cells. We show that microelectroporation provides a general method to deliver DNA, mRNA, and tRNA simultaneously. In both CHO cells and cultured neurons, microelectroporation efficiently delivers an in vitro transcribed, serine amber suppressor tRNA, leading to nonsense suppression in a mutant EGFP gene. In CHO cells, both natural and unnatural amino acids chemically appended to a suppressor tRNA are site specifically incorporated into the nicotinic acetylcholine receptor (nAChR). Electrophysiology confirms the expected functional consequences of the unnatural residue. The microelectroporation strategy described here is more general, less tedious, and less damaging to mammalian neuronal and nonneuronal cells than previous approaches to nonsense suppression in small cells and provides the first example of unnatural amino acid incorporation in mammalian cells using chemically aminoacylated tRNA.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Monahan
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D018389:Codon, Nonsense; D006224:Cricetinae; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D016153:Genes, Suppressor; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D009474:Neurons; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000596:Amino Acids; D018389:Codon, Nonsense; D008164:Luminescent Proteins; D012346:RNA, Transfer, Amino Acyl; D011956:Receptors, Cell Surface; D011978:Receptors, Nicotinic; D049452:Green Fluorescent Proteins; D000109:Acetylcholine