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1.
J Am Chem Soc ; 146(9): 5872-5882, 2024 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-38415585

RESUMEN

There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to the crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to small (a few µm or less) and thin (a few hundred nm or less) crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate coupled with rotation enables highly efficient data collection. The recorded patterns can be successfully indexed using lattice parameters obtained through 3D ED. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferentially oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies of small crystals of organic compounds.

2.
Chembiochem ; 24(21): e202300374, 2023 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-37430341

RESUMEN

With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (RF ) group adjacent to the α-carbon. RF -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. RF -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (RF -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel RF -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs).


Asunto(s)
Antineoplásicos , Péptidos de Penetración Celular , Fluorocarburos , Transporte Biológico , Péptidos de Penetración Celular/química , Aminoácidos/metabolismo
3.
Org Biomol Chem ; 20(35): 6994-7000, 2022 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-35993969

RESUMEN

N-Substituted peptides, such as peptoids and ß-peptoids, have been reported to have unique structures with diverse functions, like catalysis and manipulation of biomolecular functions. Recently, the preorganization of monomer shape by restricting bond rotations about all backbone dihedral angles has been demonstrated to be useful for de novo design of peptoid structures. Such design strategies are hitherto unexplored for ß-peptoids; to date, no preorganized ß-peptoid monomers have been reported. Here, we report the first design strategy for ß-peptoids, in which all four backbone dihedral angles (ω, ϕ, θ, ψ) are rotationally restricted on a per-residue basis. The introduction of a cyclopentane constraint realized the preorganized monomer structure and led to a ß-peptoid with a stable twisted strand shape.


Asunto(s)
Peptoides , Ciclopentanos , Péptidos/química , Peptoides/química
4.
Angew Chem Int Ed Engl ; 61(14): e202200119, 2022 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-35088931

RESUMEN

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.


Asunto(s)
Alanina , Péptidos , Alanina/análogos & derivados , Enlace de Hidrógeno , Péptidos/química , Agua/química
5.
Org Biomol Chem ; 19(47): 10326-10331, 2021 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-34821247

RESUMEN

Backbone stereochemistry of cyclic peptides has been reported to have a great influence on microsomal stability and membrane permeability, two important factors that determine oral bioavailability. Here, we comprehensively investigated the correlation between the backbone stereochemistry of cyclic hexapeptide stereoisomers and their stability in liver microsomes, as well as passive membrane permeability.


Asunto(s)
Permeabilidad de la Membrana Celular
6.
Org Biomol Chem ; 19(43): 9386-9389, 2021 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-34676842

RESUMEN

Here, we investigated the effect of CH3 to CF3 substitution on the membrane permeability of peptides. We synthesized a series of peptides with CF3 groups and corresponding nonfluorinated peptides and measured the membrane permeability of the peptides. As a result, we demonstrated that CH3 to CF3 substitution is useful for increasing the membrane permeability of di-/tri-peptides.


Asunto(s)
Péptidos
7.
J Am Chem Soc ; 142(5): 2277-2284, 2020 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-31917919

RESUMEN

Unique folded structures of natural and synthetic oligomers are the most fundamental basis for their unique functions. N-Substituted ß-peptides, or ß-peptoids, are synthetic oligomers with great potential to fold into diverse three-dimensional structures because of the existence of four rotatable bonds in a monomer with highly modular synthetic accessibility. However, the existence of the four rotatable bonds poses a challenge for conformational control of ß-peptoids. Here, we report a strategy for per-residue programming of two dihedral angles of ß-peptoids, which is useful for restricting the conformational space of the oligomers. The oligomer was found to form a unique loop conformation that is stabilized by the backbone rotational restrictions. Circular dichroism and NMR spectroscopic analyses and X-ray crystallographic analysis of the oligomer are presented. The strategy would significantly facilitate the discovery of many more unique folded structures of ß-peptoids.


Asunto(s)
Peptoides/química , Dicroismo Circular , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína
8.
J Am Chem Soc ; 141(37): 14612-14623, 2019 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-31405271

RESUMEN

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.


Asunto(s)
Peptoides/química , Agua/química , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Conformación Proteica , Teoría Cuántica
9.
Org Biomol Chem ; 17(11): 2887-2891, 2019 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-30810151

RESUMEN

Here, we report a facile permeability assay to quantitatively evaluate the membrane permeability of multiple peptides in parallel. With a fluorogenic click reaction between azidocoumarin and a terminal alkyne tag introduced on a peptide, the peptide that crossed an artificial membrane or a cell monolayer was quantitatively detected. The method allows a rapid measurement of the permeability of multiple compounds on a plate reader even in the presence of a complex mixture of biological molecules.


Asunto(s)
Permeabilidad de la Membrana Celular , Colorantes Fluorescentes/análisis , Células de Riñón Canino Madin Darby/citología , Membranas Artificiales , Imagen Óptica , Péptidos/química , Alquinos/química , Animales , Células CACO-2 , Cumarinas/química , Perros , Colorantes Fluorescentes/química , Humanos , Estructura Molecular
10.
Bioorg Med Chem Lett ; 28(3): 231-234, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29326019

RESUMEN

α-Helix-mediated protein-protein interactions (PPIs) are important targets in biological research and drug development. Peptides containing d-amino acid residues are attractive molecules for inhibiting α-helix-mediated PPIs because of their wide surface area and high protease resistance. In this study, a peptide library was constructed using a one-bead one-compound format designed to isolate left-handed α-helical peptides, which are promising molecules as inhibitors of α-helix-mediated PPIs. Screening of the library against an α-helix-mediated PPI between MDM2 and p53 yielded an inhibitor of the PPI. Design and screening of the library, and biochemical and spectroscopic studies of the discovered peptide are presented.


Asunto(s)
Biblioteca de Péptidos , Péptidos/química , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Humanos , Ligandos , Conformación Proteica en Hélice alfa , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/química , Estereoisomerismo , Proteína p53 Supresora de Tumor/química
11.
J Biol Chem ; 291(14): 7558-70, 2016 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-26851280

RESUMEN

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.


Asunto(s)
Antígenos/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Antígenos/genética , Femenino , Humanos , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Receptores de Antígenos de Linfocitos B/genética , Células Tumorales Cultivadas
12.
Bioconjug Chem ; 25(8): 1479-91, 2014 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-25073654

RESUMEN

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors.


Asunto(s)
Dextranos/química , Inmunoglobulina G/inmunología , Péptidos/química , Péptidos/inmunología , Peptidomiméticos/química , Peptidomiméticos/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Clonales , Dimerización , Células HEK293 , Humanos , Inmunoglobulina G/química , Leucemia Linfoide/patología , Ligandos , Ratones , Modelos Moleculares , Polietilenglicoles/química , Unión Proteica , Conformación Proteica
13.
Chem Sci ; 15(19): 7051-7060, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38756815

RESUMEN

Peptoids are a promising drug modality targeting disease-related proteins, but how a peptoid engages in protein binding is poorly understood. This is primarily due to a lack of high-resolution peptoid-protein complex structures and systematic physicochemical studies. Here, we present the first crystal structure of a peptoid bound to a protein, providing high-resolution structural information about how a peptoid binds to a protein. We previously reported a rigid peptoid, oligo(N-substituted alanine) (oligo-NSA), and developed an oligo-NSA-type peptoid that binds to MDM2. X-ray crystallographic analysis of the peptoid bound to MDM2 showed that the peptoid recognizes the MDM2 surface predominantly through the interaction of the N-substituents, while the main chain acts as a scaffold. Additionally, conformational, thermodynamic, and kinetic analysis of the peptoid and its derivatives with a less rigid main chain revealed that rigidification of the peptoid main chain contributes to improving the protein binding affinity. This improvement is thermodynamically attributed to an increased magnitude of the binding enthalpy change, and kinetically to an increased association rate and decreased dissociation rate. This study provides invaluable insights into the design of protein-targeting peptoids.

14.
RSC Adv ; 13(12): 8394-8397, 2023 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-36922944

RESUMEN

We compared the passive permeability of cyclosporin A (CsA) derivatives with side chain deletions across lipid bilayers. CsA maintained passive permeability after losing any one of the side chains, which suggests that the propensity of the backbone of CsA is an important component for high passive permeability.

15.
Chem Sci ; 14(2): 345-349, 2023 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-36687349

RESUMEN

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides.

16.
Nat Commun ; 14(1): 1416, 2023 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-36932083

RESUMEN

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.


Asunto(s)
Amidas , Péptidos Cíclicos , Péptidos Cíclicos/química , Metilación , Ésteres , Permeabilidad de la Membrana Celular , Péptidos/química , Permeabilidad
17.
Acc Chem Res ; 44(12): 1359-68, 2011 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-21711008

RESUMEN

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.


Asunto(s)
Evolución Molecular , ARN Catalítico/genética , ARN Catalítico/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Catálisis , Cristalografía por Rayos X , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo
18.
Angew Chem Int Ed Engl ; 51(14): 3423-7, 2012 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-22374802

RESUMEN

Designed to inhibit: by using the random nonstandard peptide integrated discovery (RaPID) system, highly potent isoform-selective inhibitors can be identified from a library of nonstandard macrocyclic peptides. These inhibitors, which contain a mechanism-based warhead residue, are active against the human deacetylase SIRT2, with IC(50) values in the low nanomolar region.


Asunto(s)
Inhibidores Enzimáticos/síntesis química , Péptidos/química , Sirtuina 2/antagonistas & inhibidores , Secuencia de Aminoácidos , Ciclización , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Cinética , Péptidos/síntesis química , Péptidos/metabolismo , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Sirtuina 2/metabolismo
19.
Chem Sci ; 12(40): 13292-13300, 2021 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-34777747

RESUMEN

The development of inhibitors of intracellular protein-protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo(N-substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo(N-substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N-substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N-substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors.

20.
J Am Chem Soc ; 130(23): 7232-4, 2008 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-18479111

RESUMEN

Here we report a new methodology for the synthesis of bicyclic peptides by using a reconstituted cell-free translation system under the reprogrammed genetic code. Cysteine (Cys) and three different nonproteinogenic amino acids, Cab, Aha, and Pgl, were simultaneously incorporated into a peptide chain. The first cyclization occurred between the chloroacetyl group of Cab and the sulfhydryl group in Cys in situ of translation, and the second cyclization on the side chains of Aha-Pgl via Cu(I)-catalyzed azide-alkyne cycloaddition was performed. This offers us a powerful means of mRNA-programmed synthesis of various peptides with uniform bicyclic scaffolds.


Asunto(s)
Péptidos Cíclicos/síntesis química , Ribosomas/química , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alquinos/química , Alquinos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Aminobutiratos/química , Aminobutiratos/metabolismo , Codón , Cisteína/química , Cisteína/metabolismo , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Moldes Genéticos
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