RESUMEN
DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.
Asunto(s)
Nanopartículas del Metal , Plata , Humanos , Cricetinae , Animales , Plata/química , Células CHO , Química Clic , Nanopartículas del Metal/química , Cricetulus , ADN/química , Insulina , Péptidos , Espectrometría de FluorescenciaRESUMEN
Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2'-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.
Asunto(s)
Oligonucleótidos , Poliaminas , ADN/química , ADN de Cadena Simple/química , Oligonucleótidos/químicaRESUMEN
Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNaseâ H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNaseâ H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNaseâ H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.
Asunto(s)
Silenciador del Gen , Nucleótidos/química , Oligonucleótidos Antisentido/química , Ribonucleasa H/metabolismo , Secuencia de Bases , Estabilidad de Enzimas , Células HeLa , Humanos , Concentración 50 Inhibidora , Desnaturalización de Ácido Nucleico , Nucleótidos/metabolismo , Oligonucleótidos Antisentido/metabolismo , ARN/química , ARN/metabolismo , Temperatura , TransfecciónRESUMEN
DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.
Asunto(s)
ADN/química , Nanotecnología/métodos , Modelos Moleculares , Nanoestructuras/química , Nanotecnología/instrumentación , Hibridación de Ácido Nucleico , Péptidos/química , Dominios ProteicosRESUMEN
Two highly specific biomolecular recognition events, nucleic acid duplex hybridization and DNA-peptide recognition in the minor groove, were coalesced in a miniature ensemble for the first time by covalently attaching a natural AT-hook peptide motif to nucleic acid duplexes via a 2'-amino-LNA scaffold. A combination of molecular dynamics simulations and ultraviolet thermal denaturation studies revealed high sequence-specific affinity of the peptide-oligonucleotide conjugates (POCs) when binding to complementary DNA strands, leveraging the bioinformation encrypted in the minor groove of DNA duplexes. The significant cooperative DNA duplex stabilization may pave the way toward further development of POCs with enhanced affinity and selectivity toward target sequences carrying peptide-binding genetic islands.
Asunto(s)
ADN/química , Oligonucleótidos/química , Péptidos/química , Sitios de Unión , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Prueba de Estudio Conceptual , Conformación ProteicaRESUMEN
Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
Asunto(s)
ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Glicina/análogos & derivados , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/antagonistas & inhibidores , ADN Bacteriano/química , ADN Superhelicoidal/química , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos Antisentido/síntesis química , Plásmidos/química , Plásmidos/metabolismo , Técnicas de Síntesis en Fase Sólida , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Mono- and diaminated 2'-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2'-amino-LNA monomers and the host oligonucleotide backbone.
Asunto(s)
Oligonucleótidos/química , ADN Complementario/metabolismo , Desoxirribonucleasas , Resistencia a Medicamentos , Estabilidad de Medicamentos , Modelos Moleculares , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , ARN Complementario/metabolismo , Electricidad EstáticaRESUMEN
The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12â nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.
Asunto(s)
Oligonucleótidos/química , Péptidos/química , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Dicroismo Circular , Cobre/química , Reacción de Cicloadición , Electroforesis en Gel de Poliacrilamida , Hibridación de Ácido Nucleico , Desnaturalización Proteica , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Galactose-modified thymidine, LNA-T, and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'-functionalized 2'-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2'-amino-LNA-T derivatives induced remarkable 3'-exonuclease resistance. These results were further investigated using molecular modeling studies.
Asunto(s)
ADN/química , Galactosa/química , Oligonucleótidos/química , Fenómenos Biofísicos , Espectroscopía de Resonancia Magnética con Carbono-13 , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , TemperaturaRESUMEN
The application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) and 2'-O-methyl-RNA (2'OMe), has improved the performance of fluorescence in situ hybridization (FISH) methods for the detection/location of clinical pathogens since they provide design versatility and thermodynamic control. However, an important limitation of FISH techniques is the low number of distinguishable targets. The use of filters in fluorescence image acquisition limits the number of fluorochromes that can be simultaneously differentiated. Recent advances in fluorescence spectral image acquisition have allowed the unambiguous identification of several microorganisms in a single sample. In this work, we aimed to combine NAM-FISH and spectral image analysis to develop and validate a new FISH variant, the spectral imaging-NAM-FISH (SI-NAM-FISH), that allows a multiplexed, robust and rapid detection of clinical pathogens. In the first stage, to implement/validate the method, we have selected seven fluorochromes with distinct spectral properties and seven bacterial species (Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter calcoaceticus). As a strong variation in fluorescence intensities is found between species and between fluorochromes, seven versions of a EUB LNA/2'OMe probe, each conjugated to one of seven fluorochromes, were used to rank species/fluorochromes by FISH and then optimize species/fluorochrome pairing. Then, final validation tests were performed using mixed populations to evaluate the potential of the technique for separating/quantifying the different targets. Overall, validation tests with different proportions of bacteria labeled with the respective fluorochrome have shown the ability of the method to correctly distinguish the species.
RESUMEN
Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC50 = 8.9 nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD50 > 5000 mg/kg and showed significant in vivo antitumor activity through promoting CD8+ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.
RESUMEN
Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.
Asunto(s)
ADN/química , Sustancias Macromoleculares/química , Simulación del Acoplamiento Molecular , Dominios Proteicos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estructura Molecular , Péptidos/química , Estructura Secundaria de Proteína , Proteínas/química , EstereoisomerismoRESUMEN
The phosphoramidite monomer of the C-nucleoside 2'-aminoethoxy-2-amino-3-methylpyridine (AE-MAP) has been synthesized for the first time and incorporated into triplex-forming oligonucleotides (TFOs). Ultraviolet melting and DNase I footprinting studies show that AE-MAP is a potent triplex-stabilizing monomer that is selective for GC base pairs. TFOs containing AE-MAP bind with high affinity to duplexes but only weakly to single stranded DNA. In addition, AE-MAP confers high nuclease resistance on oligonucleotides. TFOs containing AE-MAP have potential for gene knock-out and gene expression studies.
Asunto(s)
ADN/química , ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Oligonucleótidos/química , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Piridinas/química , Piridinas/síntesis química , Emparejamiento Base , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
2'-O-(2-Methoxyethyl)-5-(3-aminoprop-1-ynyl)-uridine phosphoramidite (MEPU) has been synthesized from d-ribose and 5-iodouracil and incorporated into triplex-forming oligonucleotides (TFOs) by automated solid-phase oligonucleotide synthesis. The TFOs gave very high triplex stability with their target duplexes as measured by ultraviolet/fluorescence melting and DNase I footprinting. The incorporation of MEPU into TFOs renders them resistant to degradation by serum nucleases.
Asunto(s)
Oligodesoxirribonucleótidos/química , Uridina/análogos & derivados , Uridina/química , Huella de ADN , Humanos , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Relación Estructura-ActividadRESUMEN
Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.
Asunto(s)
Albúminas/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , Transfección/métodos , Animales , Células Cultivadas , Femenino , Semivida , Humanos , Ratones , Ratones Endogámicos C57BL , Imagen Molecular/métodos , Oligonucleótidos Antisentido/síntesis química , Unión Proteica , Distribución TisularRESUMEN
MicroRNAs (miRNAs) are post-transcriptional gene expression regulators with potential therapeutic applications. miR-146a is a negative regulator of inflammatory processes in both tissue-resident and specialized immune cells and may therefore have therapeutic effect in inflammatory skin diseases. PepFect (PF) and NickFect (NF) type of cell-penetrating peptides (CPPs) have previously been shown to deliver miRNA mimics and/or siRNAs into cell cultures and in vivo. Here, we first demonstrate that selected PF- and NF-type of CPPs support delivery of fluorescent labelled miRNA mimics into keratinocytes (KCs) and dendritic cells (DCs). Second, we show that both PF- and NF-miR-146a nanocomplexes were equally effective in KCs, while NFs were more efficient in DCs as assessed by downregulation of miR-146a-influenced genes. None of miRNA nanocomplexes with the tested CPPs influenced the viability of KCs and DCs nor caused activation of DCs according to CD86 and CD83 markers. Transmission electron microscopy analysis with Nanogold-labelled miR-146a mimics and assessment of endocytic trafficking pathways revealed endocytosis as an active route of delivery in both KCs and DCs for all tested CPPs. However, consistent with the higher efficiency, NF-delivered miR-146a was detected more often outside endosomes in DCs. Finally, pre-injection of NF71:miR-146a nanocomplexes was confirmed to suppress inflammatory responses in a mouse model of irritant contact dermatitis as shown by reduced ear swelling response and downregulation of pro-inflammatory cytokines, including IL-6, IL-1ß, IL-33 and TNF-α. In conclusion, NF71 efficiently delivers miRNA mimics into KCs as well as DCs, and therefore may have advantage in therapeutic delivery of miRNAs in case of inflammatory skin diseases.
Asunto(s)
Péptidos de Penetración Celular , MicroARNs , Animales , Células Dendríticas , Inflamación , Queratinocitos , Ratones , MicroARNs/genéticaRESUMEN
The on-column functionalization of oligodeoxynucleotides via base-free Suzuki cross-coupling reactions is reported herein. These cross-coupling reactions were carried out with various boronic acids and either full-length modified oligonucleotides containing one or more 2'-deoxy-5-iodouridine (5IdU) monomer(s) or on oligonucleotide fragments immediately after incorporation of 5IdU. Five different functionalities were coupled to oligonucleotides containing one or three attachment points.
Asunto(s)
Oligodesoxirribonucleótidos/química , Ácidos Borónicos/química , Catálisis , Idoxuridina/análogos & derivados , Idoxuridina/química , Paladio/químicaRESUMEN
Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.
Asunto(s)
Nanopartículas/química , Oligonucleótidos/química , Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Dicroismo Circular , Modelos Moleculares , Oligonucleótidos/síntesis química , Péptidos/síntesis química , Desnaturalización Proteica , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Rayos Ultravioleta , Difracción de Rayos XRESUMEN
Incorporation of a piperazino-modified 2'-amino-LNA monomer (PipLNA-T) into oligonucleotides conferred very high affinity and base-pairing selectivity towards complementary DNA and RNA strands. Furthermore, one PipLNA-T modification provided a robust nuclease resistance that safeguarded three neighbouring natural nucleosides from 3'-exonucleolytic degradation. These favourable properties render PipLNA-T a promising oligonucleotide modification for various biological applications.