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In this Article, owing to an error during the production process, in Fig. 1a, the dark blue and light blue wedges were incorrectly labelled as 'Gâ¢C â Tâ¢A' and 'Gâ¢C â Aâ¢T', instead of 'Câ¢G â Tâ¢A' and 'Câ¢G â Aâ¢T', respectively. Fig. 1 has been corrected online.
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This corrects the article DOI: 10.1038/nchembio.2474.
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The spontaneous deamination of cytosine is a major source of transitions from Câ¢G to Tâ¢A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted Aâ¢T base pairs to Gâ¢C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of Aâ¢T to Gâ¢C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted Aâ¢T base pairs efficiently to Gâ¢C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
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Emparejamiento Base/genética , Edición Génica/métodos , Genoma Humano/genética , Adenosina Desaminasa/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , División del ADN , Células HEK293 , Humanos , Modelos Moleculares , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.
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Aminoacil-ARNt Sintetasas/metabolismo , Evolución Molecular Dirigida , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Biocatálisis , Methanocaldococcus/metabolismo , Methanosarcina/metabolismo , Conformación Molecular , Proteínas/química , Proteínas/metabolismoRESUMEN
Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution-a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.
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Aminoacil-ARNt Sintetasas/metabolismo , Lisina/análogos & derivados , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Cristalografía por Rayos X , Evolución Molecular Dirigida , Lisina/química , Lisina/metabolismo , Methanosarcina/enzimología , Modelos MolecularesRESUMEN
On-bead high-throughput screening of a medium-sized (1000-2000 Da) branched peptide boronic acid (BPBA) library consisting of 46,656 unique sequences against HIV-1 RRE RNA generated peptides with binding affinities in the low micromolar range. In particular, BPBA1 had a K(d) of 1.4 µM with RRE IIB, preference for RNA over DNA (27 fold), and selectivity of up to >75 fold against a panel of RRE IIB variants. Structure-activity studies suggest that the boronic acid moiety and "branching" in peptides are key structural features for efficient binding and selectivity for the folded RNA target. BPBA1 was efficiently taken up by HeLa and A2780 cells. RNA-footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB.
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Ácidos Borónicos/química , Sistemas de Liberación de Medicamentos , VIH-1/metabolismo , Péptidos/química , ARN Viral/metabolismo , Secuencia de Bases , VIH-1/genética , Células HeLa , Humanos , Biblioteca de Péptidos , Unión Proteica , Pliegue del ARN , Relación Estructura-Actividad , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
We report branched peptide boronic acids (BPBAs) that bind to RRE IIB from an on-bead high-throughput screening of a 3.3.4-library (46 656 compounds). We demonstrate that boronic acids are tunable moieties that afford a novel binding mode towards RNA.
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Ácidos Borónicos/química , Péptidos/química , ARN Viral/metabolismo , Secuencia de Aminoácidos , VIH-1/genética , VIH-1/metabolismo , Humanos , Biblioteca de Péptidos , Péptidos/metabolismo , Unión ProteicaRESUMEN
Rational design of RNA ligands continues to be a formidable challenge, but the potential powerful applications in biology and medicine catapults it to the forefront of chemical research. Indeed, small molecule and macromolecular intervention are attractive approaches, but selectivity and cell permeability can be a hurdle. An alternative strategy is to use molecules of intermediate molecular weight that possess large enough surface area to maximize interaction with the RNA structure but are small enough to be cell-permeable. Herein, we report the discovery of nontoxic and cell-permeable branched peptide (BP) ligands that bind to TAR RNA in the low micromolar range from on-bead high-throughput screening of 4,096 compounds. TAR is a short RNA motif in the 5'-UTR of HIV-1 that is responsible for efficient generation of full RNA transcripts. We demonstrate that BPs are selective for the native TAR RNA structure and that "branching" in peptides provides multivalent interaction, which increases binding affinity to RNA.
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Duplicado del Terminal Largo de VIH , VIH-1/metabolismo , Péptidos/metabolismo , ARN Viral/metabolismo , Regiones no Traducidas 5'/genética , Sitios de Unión , Permeabilidad de la Membrana Celular , Colorantes Fluorescentes , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/química , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Péptidos/síntesis química , Péptidos/farmacología , Unión Proteica , ARN Viral/antagonistas & inhibidores , ARN Viral/química , Coloración y Etiquetado , Relación Estructura-ActividadRESUMEN
The recognition that RNA is more than just an intermediate in the information transfer from genetic code to fully functional protein has placed it at the forefront of chemical research. RNA is important because of its vital role in regulating transcription, translation, splicing, replication and catalysis. Consequently, molecules that can bind to RNA and control its function have potential as powerful tools in biology and medicine. Herein, we report the discovery of HIV-1 TAR RNA-selective ligands using an on-bead screening of a library of 4096 branched peptides.
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Duplicado del Terminal Largo de VIH , VIH-1/genética , Biblioteca de Péptidos , Péptidos/química , ARN Mensajero/genética , ARN Viral/genética , Descubrimiento de Drogas/métodos , Péptidos/genética , Péptidos/metabolismo , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
Oxygen evolution by photosystem II (PSII) is activated by chloride and other monovalent anions. In this study, the effects of iodide on oxygen evolution activity were investigated using PSII-enriched membrane fragments from spinach. In the absence of Cl(-), the dependence of oxygen evolution activity on I(-) concentration showed activation followed by inhibition in both intact PSII and NaCl-washed PSII, which lacked the PsbP and PsbQ subunits. Using a substrate inhibition model, the range of values of the Michaelis constant K(M) in intact PSII (0.5-1.5 mM) was smaller than that in NaCl-washed PSII (1.5-5 mM), whereas values of the inhibition constant K(I) in intact PSII (9-17 mM) were larger than those in NaCl-washed PSII (1-4 mM). Studies of I(-) inhibition of Cl(-)-activated oxygen evolution in intact PSII revealed that I(-) was primarily an uncompetitive inhibitor, with uncompetitive constant K(i)' = 37 mM and Cl(-)-competitive constant K(i) > 200 mM. This result indicated that the activating Cl(-) must be bound for inhibition to take place, which is consistent with the substrate inhibition model for I(-) activation. The S(2) state multiline and g = 4.1 EPR signals in NaCl-washed PSII were examined in the presence of 3 and 25 mM NaI, corresponding to I(-)-activated and I(-)-inhibited conditions, respectively. The two S(2) state signals were observed at both I(-) concentrations, indicating that I(-) substitutes for Cl(-) in formation of the signals and that advancement to the S(2) state was not prevented by high I(-) concentrations. A model is presented that incorporates the results of this study, including the action of both chloride and iodide.