RESUMEN
This research is based on a Systematic Evolution of Ligands by EXponential enrichment, also referred to as in vitro selection against the extracellular domain of human interleukin-17 receptor A (IL-17RA). Pull-down assay via quantitative polymerase chain reaction and chemiluminescence detection showed that the cloned RNA with the enriched sequence bound to human IL-17RA and inhibited the interaction between IL-17RA and human interleukin-17A (IL-17A). We also revealed that the newly discovered IL-17RA-binding RNA aptamer bound to cellular IL-17RA, inhibited the cellular IL-17RA/IL-17A interaction, and antagonized cellular IL-17A signaling.
Asunto(s)
Interleucina-17 , Receptores de Interleucina-17 , Humanos , Receptores de Interleucina-17/química , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Interleucina-17/metabolismo , Unión ProteicaRESUMEN
Interleukin-5 (IL-5) is a type 2 cytokine involved in various allergic diseases, including severe eosinophilic asthma. In this study, we performed directed evolution against human IL-5 using systematic evolution of ligands by exponential enrichment (SELEX) from multiple mRNA-displayed peptide libraries. Peptide libraries were prepared with Escherichia coli-based reconstituted cell-free transcription and translation coupling system (PURE system) and spontaneously cyclized using multiple intramolecularly thiol-reactive benzoic acid-derived linkers, which were ribosomally incorporated through genetic code expansion. We successfully identified multiple novel IL-5-binding unnatural cyclic peptides with different cyclization linkers from multiple highly diverse mRNA-displayed libraries. Chemical dimerization was also performed to increase the avidity of unnatural cyclic IL-5-binding peptides. The novel IL-5-binding unnatural cyclic peptides discovered in this study could be used in various research, therapeutic, and diagnostic applications involving IL-5 signaling.
Asunto(s)
Biblioteca de Péptidos , Péptidos Cíclicos , Escherichia coli/genética , Código Genético , Humanos , Interleucina-5/genética , Péptidos Cíclicos/genética , ARN Mensajero/genéticaRESUMEN
Interaction between the pro-inflammatory cytokine interleukin-23 (IL-23) and IL-23 receptor (IL-23R) is related to the development of inflammatory autoimmune diseases such as psoriasis, inflammatory bowel disease, and Crohn's disease. In this study, we conducted systematic evolution of ligands by exponential enrichment (SELEX) for in vitro selection against human IL-23 and observed RNA sequence enrichment in the final SELEX round. IL-23-pull-down assay by chemiluminescence detection and fluorescence imaging demonstrated that SELEX-enriched RNA clone bound to IL-23. Quantitative polymerase chain reaction-based pull-down assay using the IL-23 alpha (IL-23A) subunit, a component of the IL-23 heterodimer, indicated that the RNA clone bound to IL-23A, which is favorable for autoimmune disease treatment. We also observed that the novel IL-23-binding RNA aptamer inhibited interaction between IL-23 and IL-23R. Thus, the novel IL-23-binding RNA aptamer can be used for IL-23 studies and has potential to be used for IL-23 diagnosis and IL-23-related inflammatory autoimmune disease treatment.
Asunto(s)
Aptámeros de Nucleótidos , Enfermedades Autoinmunes , Aptámeros de Nucleótidos/metabolismo , Humanos , Interleucina-23 , Ligandos , ARN , Técnica SELEX de Producción de Aptámeros/métodosRESUMEN
The interaction of the multifunctional cytokine interleukin (IL)-6 and its receptor (IL-6R) is involved in various diseases, including not only autoimmune diseases such as rheumatoid arthritis but also cancer and cytokine storms in coronavirus disease 2019 (COVID-19). In this study, systematic evolution of ligands by exponential enrichment (SELEX) against human IL-6R from mRNA-displayed unnatural peptide library ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid (mClPh) incorporated by genetic code expansion (sense suppression) was performed using the PURE (Protein synthesis Using Recombinant Elements) system. A novel 13-mer unnatural mClPh-cyclized peptide that binds to the extracellular domain of IL-6R was discovered from an extremely diverse random peptide library. In vitro affinity maturation of IL-6R-binding unnatural mClPh-cyclized peptide from focused libraries was performed, identifying two IL-6R-binding unnatural mClPh-cyclized peptides by next-generation sequencing. Because cyclization can increase the protease resistance of peptides, novel IL-6R-binding mClPh-cyclized peptides discovered in this study have the potential to be used for a variety of research, therapeutic, and diagnostic applications involving IL-6/IL-6R signaling.
Asunto(s)
Ácido Benzoico/química , Péptidos/química , Receptores de Interleucina-6/química , Ribosomas/química , Ciclización , Código Genético , Humanos , Biblioteca de Péptidos , ARN Mensajero , Técnica SELEX de Producción de AptámerosRESUMEN
The interaction of human epidermal growth factor receptor 3 (HER3) and heregulin (HRG) is involved in resistance to human epidermal growth factor receptor 2 (HER2)-targeted cancer treatment, such as therapies using anti-HER2 monoclonal antibody. Therefore, inhibition of the HER3/HRG interaction is potentially valuable therapeutic target for cancer treatment. In this study, we used in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX) against the extracellular domain of human HER3, and discovered a novel RNA aptamer. Pull-down and bio-layer interferometry assays showed that RNA aptamer discovered specifically bound to HER3 with a dissociation constant (KD) of 700 nM. Pull-down assays using chemiluminescence detection also revealed that the HER3-binding RNA aptamer inhibited interactions between HER3 and human HRG. These results indicated that the novel HER3-binding RNA aptamer has potential to be used as basic tool in a range of applications involving HER3/HRG interactions, including research, therapeutic, and diagnostic applications.
Asunto(s)
Aptámeros de Nucleótidos/análisis , Receptores ErbB/antagonistas & inhibidores , Neurregulina-1/antagonistas & inhibidores , Neurregulina-1/metabolismo , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/metabolismo , Técnica SELEX de Producción de Aptámeros , Aptámeros de Nucleótidos/aislamiento & purificación , Secuencia de Bases , Receptores ErbB/metabolismo , Humanos , Cinética , Luminiscencia , Unión Proteica/efectos de los fármacos , Receptor ErbB-3/químicaRESUMEN
Tumor necrosis factor-alpha (TNFα) is a multifunctional cytokine associated with inflammation, immune responses, and autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. In the present study, we performed in vitro selection, systematic evolution of ligands by exponential enrichment (SELEX) against human TNFα from mRNA-displayed peptide library prepared with Escherichia coli-reconstituted cell-free transcription/translation system (PURE system) and cyclized by N-chloroacetyl-N-methyl-d-phenylalanine incorporated by genetic code expansion (sense suppression). We identified a novel TNFα-binding thioether-cyclized peptide that contains an N-methyl-d-phenylalanine. Since cyclic structure and presence of an N-methyl-d-amino acid can increase proteolytic stability, the TNFα binding peptide has potential to be used for therapeutic, research and diagnostic applications.
Asunto(s)
Biblioteca de Péptidos , Péptidos Cíclicos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Código Genético , Humanos , Metilación , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Fenilalanina/análogos & derivados , Fenilalanina/genética , Unión Proteica , ARN Mensajero/genética , Técnica SELEX de Producción de AptámerosRESUMEN
Interleukin-6 (IL-6) binds to the IL-6 receptor (IL-6R) subunit, related to autoimmune diseases and cytokine storm in COVID-19. In this study, we performed systematic evolution of ligands by exponential enrichment and identified a novel RNA aptamer. This RNA aptamer not only bound to IL-6R with a dissociation constant of 200 n m, but also inhibited the interaction of IL-6R with IL-6.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Interleucina-6/antagonistas & inhibidores , Receptores de Interleucina-6/antagonistas & inhibidores , Aptámeros de Nucleótidos/química , Secuencia de Bases , COVID-19/complicaciones , Síndrome de Liberación de Citoquinas/etiología , ADN Viral/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Técnica SELEX de Producción de AptámerosRESUMEN
We report novel, ribosomally incorporatable, and intramolecularly cysteine-reactive fluorobenzoic acid-derived linkers for SELEX of mRNA-displayed unnatural peptides, which spontaneously cyclized via intramolecular nucleophilic aromatic substitutions forming thioethers. With this strategy we identified several novel PCSK9-binding peptides.