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1.
ACS Synth Biol ; 13(7): 2141-2149, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38904157

RESUMO

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.


Assuntos
Aminoácidos , Evolução Molecular Direcionada , Escherichia coli , Mutagênese , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Aminoácidos/genética , Aminoácidos/metabolismo , Células HEK293 , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo
2.
Angew Chem Int Ed Engl ; 63(9): e202316428, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38279536

RESUMO

Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus-assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base-pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl ), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell-lines for ncAA mutagenesis.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Escherichia coli/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Mutagênese
3.
Bioconjug Chem ; 35(1): 64-71, 2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-38103182

RESUMO

The ability to engineer adeno-associated virus (AAV) vectors for targeted transduction of specific cell types is critically important to fully harness their potential for human gene therapy. A promising approach to achieve this objective involves chemically attaching retargeting ligands onto the virus capsid. Site-specific incorporation of a bioorthogonal noncanonical amino acid (ncAA) into the AAV capsid proteins provides a particularly attractive strategy to introduce such modifications with exquisite precision. In this study, we show that using ncAA mutagenesis, it is possible to systematically alter the attachment site of a retargeting ligand (cyclic-RGD) on the AAV capsid to create diverse conjugate architectures and that the site of attachment heavily impacts the retargeting efficiency. We further demonstrate that the performance of these AAV conjugates is highly sensitive to the stoichiometry of capsid labeling (labels per capsid), with an intermediate labeling density providing optimal activity for cRGD-mediated retargeting. Finally, we developed a technology to more precisely control the number of attachment sites per AAV capsid by selectively incorporating an ncAA into the minor capsid proteins with high fidelity and efficiency, such that AAV conjugates with varying stoichiometry can be synthesized. Together, this platform provides unparalleled control over the site and stoichiometry of capsid modification, which will enable the development of next-generation AAV vectors tailored with desirable attributes.


Assuntos
Proteínas do Capsídeo , Capsídeo , Humanos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Capsídeo/química , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos , Transdução Genética
4.
Nat Methods ; 20(1): 95-103, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36550276

RESUMO

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus.


Assuntos
Aminoacil-tRNA Sintetases , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo
5.
ACS Chem Biol ; 15(6): 1535-1540, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32330002

RESUMO

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.


Assuntos
Código Genético , Selenocisteína/química , Selenoproteínas/genética , Códon de Terminação , Escherichia coli/genética , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Leucina-tRNA Ligase/química , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Selenoproteínas/química
6.
Curr Opin Chem Biol ; 46: 164-171, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30086446

RESUMO

How a virus efficiently invades its host cell and masterfully engineers its properties provides valuable lessons and resources for the emerging discipline of synthetic biology, which seeks to create engineered biological systems with novel functions. Recently, the toolbox of synthetic biology has also been enriched by the genetic code expansion technology, which has provided access to a large assortment of unnatural amino acids with novel chemical functionalities that can be site-specifically incorporated into proteins in living cells. The synergistic interplay of these two disciplines holds much promise to advance their individual progress, while creating new paradigms for synthetic biology. In this review we seek to provide an account of the recent advances at the interface of these two research areas.


Assuntos
Aminoácidos/genética , Código Genético , Engenharia Genética/métodos , Biologia Sintética/métodos , Vírus/genética , Animais , Humanos , Engenharia de Proteínas , Proteínas/genética
7.
Methods Mol Biol ; 1728: 313-326, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29405007

RESUMO

The ability to modify the capsid proteins of human viruses is desirable both for installing probes to study their structure and function, and to attach retargeting agents to engineer viral infectivity. However, the installation of such capsid modifications currently faces two major challenges: (1) The complex and delicate capsid proteins often do not tolerate large modifications, and (2) capsid proteins are composed of the 20 canonical amino acids, precluding site-specific chemical modification of the virus. Here, we describe a technology for generating adeno-associated virus (AAV) while incorporating an unnatural amino acid (UAA) into specific sites of the virus capsid. Incorporation of this UAA is generally tolerated well, presumably due to its small structural footprint. The resulting virus can be precisely functionalized at the site of UAA incorporation using chemoselective conjugation strategies targeted toward the azido side chain of this UAA. This technology provides a powerful way to modify AAV with unprecedented precision to both probe and engineer its entry process.


Assuntos
Aminoácidos/genética , Proteínas do Capsídeo/genética , Códon , Dependovirus/genética , Vetores Genéticos/genética , Aminoácidos/química , Proteínas do Capsídeo/química , Cromatografia de Afinidade , Dependovirus/isolamento & purificação , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Código Genético , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/genética
8.
Biochem Soc Trans ; 45(2): 555-562, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28408495

RESUMO

In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology.


Assuntos
Aminoácidos/genética , Mamíferos/genética , Engenharia de Proteínas/métodos , Animais , Código Genético , Humanos , Mutagênese , Proteínas/química
9.
Angew Chem Int Ed Engl ; 56(15): 4234-4237, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28294501

RESUMO

Viruses utilize distinct binding interactions with a variety of host factors to gain entry into host cells. A chemical strategy is described to precisely perturb a specific molecular interaction between adeno-associated virus and its host cell, which can be rapidly reversed by light. This strategy enables pausing the virus entry process at a specific stage and then restart it rapidly with a non-invasive stimulus. The ability to synchronize the invading virus population at a discrete step in its entry pathway will be highly valuable for enabling facile experimental characterization of the molecular processes underlying this process. Additionally, adeno-associated virus has demonstrated outstanding potential for human gene therapy. This work further provides a potential approach to create therapeutic vectors that can be photoactivated in vivo with high spatial and temporal control.


Assuntos
Dependovirus/química , Interações entre Hospedeiro e Microrganismos , Terapia Genética , Vetores Genéticos/química , Humanos , Processos Fotoquímicos
10.
Angew Chem Int Ed Engl ; 55(36): 10645-9, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27483453

RESUMO

The ability to target the adeno-associated virus (AAV) to specific types of cells, by altering the cell-surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor-targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv ß3 integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site-dependent, underscoring the importance of achieving site-selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity.


Assuntos
Aminoácidos/química , Capsídeo/química , Dependovirus/química , Peptídeos Cíclicos/química , Aminoácidos/metabolismo , Capsídeo/metabolismo , Linhagem Celular Tumoral , Dependovirus/fisiologia , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Integrina alfaVbeta3/metabolismo , Modelos Moleculares , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Peptídeos Cíclicos/metabolismo , Internalização do Vírus
11.
Tetrahedron Lett ; 56(23): 3365-3367, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-26028781

RESUMO

The direct, regioselective, and stereoselective arylation of activated alkynes with aryl iodides using a nickel catalyst and manganese reductant is described. The reaction conditions are mild (40 °C in MeOH, no acid or base) and an intermediate organomanganese reagent is unlikely. Functional groups tolerated include halides and pseudohalides, free and protected anilines, and a benzyl alcohol. Other activated alkynes including an amide and a ketone also reacted to form arylated products in good yields.

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