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Abstract: Although prostaglandin I2 is used to treat pulmonary hypertension (PH), continuous intravenous administration is necessary. We investigated whether human PGIS (hPGIS) gene transfer using adeno-associated virus (AAV) vector was effective in treating an animal model of PH. PH was induced by subjecting mice to 10% O2. Type 1-AAV-hPGIS was injected into the left thigh muscle after 24h. Significant PH was induced at 8 weeks, but AAV-hPGIS administration significantly inhibited the increase in RV systolic pressure. PH-induced BNP up-regulation in the RV was reduced to the control level. The severe medial thickening of pulmonary arteries in PH was significantly suppressed by AAV-hPGIS. The hPGIS gene was detected only on the injected side. No pathological changes were observed at the injected site. At 24 weeks, all PH mice were deceased, but 47% of AAV-hPGIS-treated mice survived. This study demonstrated that AAV-hPGIS administration was effective in treating PH and prolonging survival. [Copyright &y& Elsevier]

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. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • Novel interleukin 23 (IL-23)-binding RNA aptamer was discovered by using the SELEX. • The discovered RNA aptamer inhibited the interaction between IL-23 and IL-23R. [ABSTRACT FROM AUTHOR]

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. • mRNA-displayed peptide libraries were cyclized with multiple benzoate-type linkers. • Benzoate-derived linkers were ribosomally incorporated by genetic code expansion. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • Multiple novel IL-5-binding unnatural thioether-cyclized peptides were discovered. • Chemical dimerization of IL-5-binding unnatural cyclic peptides was performed. [ABSTRACT FROM AUTHOR]

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 (K D) 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. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • Novel anti-human epidermal growth factor receptor 3 (HER3) aptamer was discovered. • The discovered RNA aptamer inhibits the interaction between HER3 and heregulin. [ABSTRACT FROM AUTHOR]

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. • m-(Chloromethyl)benzoic acid was ribosomally incorporated by genetic code expansion. • mRNA-displayed peptide library was cyclized with m-(chloromethyl)benzoic acid. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • A novel IL-6R-binding unnatural thioether-cyclized peptide was discovered. • In vitro affinity maturation of IL-6R-binding unnatural cyclic peptide was performed. [ABSTRACT FROM AUTHOR]

Protein labeling with a functional molecule is a technique widely used for protein research. The covalent reaction of self-labeling peptide tags with synthetic probe–modified small molecules enables tag-fused protein labeling with chemically diverse molecules, including fluorescent probes. We report the discovery, by in vitro directed evolution, of a novel 23-mer dibenzocyclooctyne (DBCO)-reactive peptide (DRP) tag using Systematic Evolution of Ligands by EXponential enrichment (SELEX) with a combination of a reconstituted cell-free translation system (PURE system) and cDNA display. The N- and C-terminal DRP truncations created a shorter 16-mer DBCO-reactive peptide (sDRP) tag without significant reactivity reduction. By fusing the sDRP tag to a model protein, we showed the chemical labeling and in-gel fluorescence imaging of the sDRP-fused protein using a fluorescent DBCO probe. Results showed that sDRP tag–mediated protein labeling has potential for use as a basic molecular tool in a variety of applications for protein research. • Highly diverse cDNA-displayed peptide tag library was constructed with PURE system. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • Novel dibenzocyclooctyne (DBCO)-reactive short peptide tags were discovered. • DBCO-reactive peptide tag-fused protein was labeled with a fluorescent DBCO probe. [ABSTRACT FROM AUTHOR]

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. • Reconstituted cell-free transcription/translation system (PURE system) was prepared. • Chloroacetyl N -methyl- d -phenylalanine was incorporated by genetic code expansion. • mRNA-displayed unnatural cyclic peptide having N -methyl- d -amino acid was prepared. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • A novel TNFα-binding cyclic peptide having N -methyl- d -amino acid was discovered. [ABSTRACT FROM AUTHOR]