RESUMEN
We developed a photoreactive molecular glue, BPGlue-N3, which can provide a universal strategy to enhance the efficacy of DNA aptamers by temporary-to-permanent stepwise stabilization of their conjugates with target proteins. As a proof-of-concept study, we applied BPGlue-N3 to the SL1 (DNA aptamer)/c-Met (target protein) conjugate system. BPGlue-N3 can adhere to and temporarily stabilize this aptamer/protein conjugate multivalently using its guanidinium ion (Gu+) pendants that form a salt bridge with oxyanionic moieties (e.g., carboxylate and phosphate) and benzophenone (BP) group that is highly affinitive to DNA duplexes. BPGlue-N3 is designed to carry a dual-mode photoreactivity; upon exposure to UV light, the temporarily stabilized aptamer/protein conjugate reacts with the photoexcited BP unit of adhering BPGlue-N3 and also a nitrene species, possibly generated by the BP-to-N3 energy transfer in BPGlue-N3. We confirmed that SL1, covalently conjugated with c-Met, hampered the binding of hepatocyte growth factor (HGF) onto c-Met, even when the SL1/c-Met conjugate was rinsed prior to the treatment with HGF, and suppressed cell migration caused by HGF-induced c-Met phosphorylation.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Aptámeros de Nucleótidos/química , Azidas/química , Benzofenonas/química , Línea Celular Tumoral , Movimiento Celular , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Microscopía Confocal , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-met/química , Rayos UltravioletaRESUMEN
Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/agonistas , Receptores de Citocinas/agonistas , Células A549 , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Movimiento Celular/efectos de los fármacos , Dimerización , Factor de Crecimiento de Hepatocito/agonistas , Factor de Crecimiento de Hepatocito/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Microscopía Fluorescente , Unión Proteica , Proteínas Proto-Oncogénicas c-met/agonistas , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling. We used 5-Formyl-2'-deoxyuridine as a model compound and demonstrated that chemical labeling dramatically alters its molecular conductance compared with that of canonical nucleotides; thus, facilitating statistical discrimination, which is impeded in the unlabeled state. This work introduces a chemical strategy that overcomes the intrinsic difficulty in quantum-tunneling-based modification analysis-the similarity of the molecular conductance of the nucleobases of interest.
Asunto(s)
ADN/análisis , Desoxiuridina/análogos & derivados , Teoría Cuántica , Desoxiuridina/química , Conductividad Eléctrica , Estructura MolecularRESUMEN
We developed a dendritic molecular glue PCGlue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PCGlue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/farmacología , Dendrímeros/farmacología , Guanidinas/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/efectos de la radiación , Línea Celular Tumoral , Dendrímeros/síntesis química , Dendrímeros/efectos de la radiación , Guanidinas/síntesis química , Guanidinas/efectos de la radiación , Factor de Crecimiento de Hepatocito/química , Humanos , Unión Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-met/química , Rayos UltravioletaRESUMEN
The reprogramming of receptor-ligand interactions affords an opportunity to direct cells to respond to user-defined external cues. Although this has often been achieved via the genetic engineering of receptors, an alternative, nongenetic approach is highly demanded. In this article, we propose the design of oligonucleotide-based synthetic switches that feature the ability to reprogram the ligand specificity of the growth factor receptor. We demonstrated that our synthetic switches induced growth factor signaling via the formation of the dynamic complex with specific external cues that would otherwise not induce the signaling. This chemical approach may be applied to designing a new class of chemical tools that can control the activities of native cells and represent smart and safer regenerative drugs.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/farmacología , Ligandos , Oligonucleótidos/metabolismo , Oligonucleótidos/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Aptámeros de Nucleótidos/química , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Oligonucleótidos/química , Proteínas Tirosina Quinasas Receptoras/química , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Oligonucleotide-based hepatocyte growth factor (HGF) mimetics are described. A DNA aptamer to Met, a cognate receptor for HGF, was shown to induce Met activation when used in dimer form. The most potent aptamer dimer, ss-0, which was composed solely of 100-mer single-stranded DNA, exhibited nanomolar potency. Aptamer ss-0 reproduced HGF-induced cellular behaviors, including migration and proliferation. The present work sheds light on oligonucleotides as a novel chemical entity for the design of growth factor mimetics.
Asunto(s)
Factor de Crecimiento de Hepatocito/química , Imitación Molecular , Oligonucleótidos/química , Secuencia de Bases , Línea Celular , Dimerización , Humanos , Oligonucleótidos/genética , Fosforilación , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
DNA aptamers have attracted attention as an alternative modality for biomolecules due to their excellent target binding specificity and thermal stability, and they are also expected to be applied as artificial agonists for receptor proteins. DNA aptamer agonist TD0 targeting the receptor of fibroblast growth factor (FGFR), which plays an important role in the fields of wound healing and regenerative medicine, has been reported to induce cellular responses as well as its native ligands. However, it was also noted that there were some different responses upon long-term stimulation, suggesting that the intracellular signals induced by DNA aptamer agonist TD0 are different from those of natural ligands. In this paper, we comprehensively analyzed the intracellular signals induced by DNA aptamer agonist TD0 targeting FGFR1, and compared them with those by natural protein ligand FGF2. It was found that the intracellular signals were highly similar for short-term stimulation. On the other hand, the receptor and the downstream cellular signals showed different activation behaviors for long-time stimulation. Evaluating the stability and sustained activity of DNA aptamer agonist TD0 and FGF2 in the medium suggested that ligand stability may be important in properly regulating cellular responses.
RESUMEN
Postoperative peritoneal adhesion (PPA) is a prevalent complication of abdominal surgery, posing a significant hindrance to postsurgical recovery. Although several strategies have been developed to alleviate and prevent adhesions, their efficacy remains unsatisfactory. For the first time, we studied the therapeutic effect and mechanism of our recently developed thermally stable oligonucleotide-based mimetics of hepatocyte growth factor (HGF DNA aptamer) to prevent PPA. The HGF DNA aptamer effectively inhibited canonical TGF-ß1 signaling transduction, partially suppressing mesothelial mesenchymal transition. Additionally, the aptamer, respectively, upregulated and downregulated the expression of tissue plasminogen activator and plasminogen activator inhibitor 1, thereby enhancing fibrinolytic activity. As a pleiotropic factor, the HGF DNA aptamer also enhanced the migratory and proliferative capacities of mesothelial cells. Finally, the aptamer demonstrated a higher level of effectiveness in preventing PPAs than the commercially available antiperitoneal adhesion barrier, Seprafilm. Due to its therapeutic benefits, excellent stability, biosafety, cost-effectiveness, and versatility, the HGF DNA aptamer demonstrates promise for preventing PPA in future clinical settings.
Asunto(s)
Aptámeros de Nucleótidos , Transición Epitelial-Mesenquimal , Fibrinólisis , Factor de Crecimiento de Hepatocito , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Adherencias Tisulares/prevención & control , Humanos , Fibrinólisis/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Ensayo de Materiales , Tamaño de la Partícula , Complicaciones Posoperatorias/prevención & controlRESUMEN
Recently, targeted protein degradation (TPD) has attracted much attention as a powerful strategy for effective inhibition of disease-related proteins. However, development of ligands with high affinity and specificity for a target protein is still a demanding task and poses a particular challenge for designing TPD therapeutics. In this work, we report a novel TPD strategy called aptamer-mediated cleavage of extracellular antigen (Apt-clean), where oligonucleotide-based affinity agents are used for selective recruitment of proteases to target membrane proteins. Our data demonstrate that Apt-clean induces selective degradation of the target protein both in vitro and in cellulo. In addition, the potential of Apt-clean was demonstrated through the inhibition of tumor-related growth factor signaling. This novel TPD modality may serve as an efficient and flexible strategy for targeting membrane proteins.
Asunto(s)
Aptámeros de Nucleótidos , Proteínas de la Membrana , Línea Celular Tumoral , Transducción de SeñalRESUMEN
Cell membrane receptors regulate cellular responses through sensing extracellular environmental signals and subsequently transducing them. Receptor engineering provides a means of directing cells to react to a designated external cue and exert programmed functions. However, rational design and precise modulation of receptor signaling activity remain challenging. Here, we report an aptamer-based signal transduction system and its applications in controlling and customizing the functions of engineered receptors. A previously reported membrane receptor-aptamer pair was used to design a synthetic receptor system that transduces cell signaling depending on exogenous aptamer input. To eliminate the cross-reactivity of the receptor with its native ligand, the extracellular domain of the receptor was engineered to ensure that the receptor was solely activated by the DNA aptamer. The present system features tunability in the signaling output level using aptamer ligands with different receptor dimerization propensities. In addition, the functional programmability of DNA aptamers enables the modular sensing of extracellular molecules without the need for genetic engineering of the receptor.
Asunto(s)
Aptámeros de Nucleótidos , Receptores Artificiales , Aptámeros de Nucleótidos/genética , Receptores de Superficie Celular , Ligandos , Transducción de Señal/fisiologíaRESUMEN
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 , PermeabilidadRESUMEN
Dopamine (DA) is a monoamine neurotransmitter that plays important roles in the brain, and whose levels in the brain are associated with several neurological and psychiatric disorders. Therefore, DA metabolism inhibitors have been used as therapeutic agents. Here, we report a (1)H NMR probe for the in situ analysis of DA metabolism, and its application to DA inhibitor screening. We designed doubly (13)C-labeled DA ((13)C(2)-DA) as the probe. The combination of the (13)C(2)-DA and (1)H-{(13)C-(13)C'} NMR technique allowed the selective and thus in situ monitoring of DA metabolism. Using (13)C(2)-DA, we successfully measured the efficacies of different inhibitors in a tissue sample, allowing us to improve the in situ inhibitory efficacy of the known DA metabolism inhibitor, clorgyline.
Asunto(s)
Dopaminérgicos/metabolismo , Antagonistas de Dopamina/farmacología , Dopamina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Espectroscopía de Resonancia Magnética/métodos , Animales , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Dopamina/química , Dopaminérgicos/química , Hígado/metabolismo , Ratones , Monoaminooxidasa/metabolismoRESUMEN
Monoamine oxidase (MAO) A is a flavoenzyme that catalyzes the oxidation of biologically important monoamines and is thought to be associated with psychiatric disorders. Here, we report a strategy for rationally designing a (19)F magnetic resonance imaging probe for the specific detection of human MAO-A (hMAO-A) activity. Our designed (19)F probe was oxidized expeditiously by hMAO-A to produce 2-fluoro-4-nitrophenol via a spontaneous ß-elimination mechanism. Concomitant with the structural change of the probe to the product, the (19)F chemical shift changed by 4.2 ppm, which was enough to visualize the probe and enzymatic product separately. Importantly, our probe achieved excellent discrimination of hMAO-A from its isoform hMAO-B.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Sondas Moleculares/química , Monoaminooxidasa/análisis , Radioisótopos de Flúor/química , Humanos , Monoaminooxidasa/química , Nitrofenoles/química , Conformación ProteicaRESUMEN
Cell culture media are often supplemented with recombinant growth factors and cytokines to reproduce biological conditions in vitro. Basic fibroblast growth factor (bFGF) has been widely used to support the pluripotency and self-renewal activity of human induced pluripotent stem cells (hiPSCs). We had previously developed a synthetic surrogate for bFGF on the basis of a DNA aptamer that binds to one of the FGF receptors. Since DNA aptamers have advantages over recombinant proteins in terms of thermal stability and production cost, replacing recombinant growth factors in cell culture media with DNA aptamers would be of great interest. Herein, we describe our protocol for feeder-free hiPSC culture using a DNA aptamer-based mimic of bFGF.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Imitación Molecular , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, the aberrant activity of the receptors has been associated with cancer malignancy. One of the main causes of the aberrant receptor activation is the overexpression of receptors and the resultant formation of unliganded receptor dimers, which can be activated in the absence of external ligand molecules. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that specifically binds to fibroblast growth factor receptor 2b and inhibits the aberrant receptor activation and signaling. Our investigation suggests that this aptamer inhibits the formation of the receptor dimer occurring in the absence of external ligand molecules. This work presents a new inhibitory function of aptamers and the possibility of oligonucleotide-based therapeutics for cancer.
RESUMEN
Optochemical tools that can modulate the activity of the target protein provide an opportunity for studying and regulating the related biological processes. Here we present a DNA-based nongenetic optochemical tool that can control the dynamics of growth factor signaling. This photo-caged mimicry of growth factor can be a promising tool for elucidating a linkage between the dynamics of signaling and the resulting biological outcomes, as well as for manipulating cellular functions and the fate of living cells.
Asunto(s)
ADN/metabolismo , Proteínas/metabolismo , Animales , ADN/química , Fenómenos Ópticos , Células PC12 , Proteínas/química , Ratas , Transducción de Señal , Rayos UltravioletaRESUMEN
A quantum sequencer offers a scalable electrical platform for single-molecule analysis of genomic events. A thymidine (dT) analog exhibiting uniquely high single-molecule conductance is a key element in capturing DNA synthesis dynamics by serving as a decodable marker for enzymatic labeling of nascent strands. However, the current design strategies of dT analogs that focus on their molecular orbital energy levels require bulky chemical modifications to extend the π-conjugation, which hinders polymerase recognition. We report herein a polymerase-compatible dT analog that is highly identifiable in quantum sequencing. An ethynyl group is introduced as a small gold-binding motif to differentiate the nucleobase-gold electronic coupling, which has been an overlooked factor in modifying nucleobase conductance. The resulting C5-ethynyl-2'-deoxyuridine exhibits characteristic signal profiles that allowed its correct identification at a 93% rate while maintaining polymerase compatibility. This study would expand the applicability of quantum sequencing by demonstrating a robust nucleoside marker with high identifiability.
RESUMEN
Although growth factors have great therapeutic potential because of their regenerative functions, they often have intrinsic drawbacks, such as low thermal stability and high production cost. Oligonucleotides have recently emerged as promising chemical entities for designing synthetic alternatives to growth factors. However, their applications in vivo have been recognized as a challenge because of their susceptibility to nucleases and limited distribution to a target tissue. Here, we present the first example of oligonucleotide-based growth factor mimetics that exerts therapeutic effects at a target tissue after systemic injection. The aptamer was designed to dimerize a growth factor receptor for its activation and mitigated the progression of Fas-induced fulminant hepatitis in a mouse model. This unprecedented functionality of the aptamer can be reasonably explained by its high nuclease stability and migration to the liver parenchyma. These mechanistic analyses provided insights for the successful application of aptamer-based receptor agonists.
Asunto(s)
Aptámeros de Nucleótidos , ADN/farmacología , Péptidos y Proteínas de Señalización Intercelular/agonistas , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , ADN/química , ADN/uso terapéutico , Desoxirribonucleasas/metabolismo , Estabilidad de Medicamentos , Modelos Biológicos , Conformación de Ácido Nucleico , Unión Proteica , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismoRESUMEN
Replacing expensive and thermally unstable growth factors with synthetic alternatives has been an important issue in stem cell-based regenerative medicines. Here we developed DNA aptamer-assemblies that act as functional mimics of basic fibroblast growth factor (bFGF), one of the essential factors for stem cell culture. The most potent aptamer assembly named TD0, composed solely of 76-mer single-stranded DNA, could support the self-renewal and pluripotency of induced pluripotent stem cells (iPSCs). This work presents the first application of DNA aptamer in the maintenance of iPSCs.
Asunto(s)
Aptámeros de Nucleótidos/química , Materiales Biocompatibles/química , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/farmacología , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosforilación , Unión Proteica , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/agonistas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Quantum-tunneling-based DNA sequencing is a single molecular technology that has great potential for achieving facile and high-throughput DNA sequencing. In principle, the sequence of DNA could be read out by the time trace of the tunnel current that can be changed according to molecular conductance of nucleobases passing through nanosized gap electrodes. However, efficient base-calling of four genetic alphabets has been seriously impeded due to the similarity of molecular conductance among canonical nucleotides. In this article, we demonstrate that replacement of canonical 2'-deoxyadenosine (dA) with a highly conductive dA analogue, 7-deaza dA, could expand the difference of molecular conductance between four genetic alphabets. Additionally, systematic evaluation of molecular conductance using a series of dA and dG analogues revealed that molecular conductance of the nucleotide is highly dependent on the HOMO level. Thus, the present study demonstrating that signal characteristics of the nucleotide can be modulated based on the HOMO level provides a widely applicable chemical approach and insight for facilitation of single molecular sensing as well as DNA sequencing based on quantum tunneling.