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Development of mammalian cell logic gates controlled by unnatural amino acids.

ABSTRACT: Mammalian cell logic gates hold great potential for wide-ranging applications. However, most of those currently available are controlled by drug(-like) molecules with inherent biological activities. To construct truly orthogonal circuits and artificial regulatory pathways, biologically inert molecules are ideal molecular switches. Here, we applied genetic code expansion and engineered logic gates controlled by two biologically inert unnatural amino acids. Genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase/tRNA pairs for co-translational and site-specific unnatural amino acid incorporation conventionally in response to an amber (UAG) codon. By screening 11 quadruplet-decoding pyrrolysyl tRNA variants from the literature, we found that all variants decoding CUAG or AGGA tested here are functional in mammalian cells. Using a quadruplet-decoding orthogonal pair together with an amber-decoding pair, we constructed logic gates that can be successfully controlled by two different unnatural amino acids, expanding the scope of genetic code expansion and mammalian cell logic circuits.

SUBMITTER: Mills EM

PROVIDER: S-EPMC9017196 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Development of mammalian cell logic gates controlled by unnatural amino acids.

Mills Emily M EM Barlow Victoria L VL Jones Arwyn T AT Tsai Yu-Hsuan YH

Cell reports methods 20210916 6

Mammalian cell logic gates hold great potential for wide-ranging applications. However, most of those currently available are controlled by drug(-like) molecules with inherent biological activities. To construct truly orthogonal circuits and artificial regulatory pathways, biologically inert molecules are ideal molecular switches. Here, we applied genetic code expansion and engineered logic gates controlled by two biologically inert unnatural amino acids. Genetic code expansion relies on orthogo ...[more]

PMID: 35474893

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Project description:A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5-10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F'-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F'-[K(DipR)5]) alone and F'-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F'-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F'-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F'-[K(DipR)5] alone or F'-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

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Development of mammalian cell logic gates controlled by unnatural amino acids.

Publications

Development of mammalian cell logic gates controlled by unnatural amino acids.

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