title: Calcium Influx of Mast Cells Is Inhibited by Aptamers Targeting the First Extracellular Domain of Orai1.
abstract: Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we identified oligonucleotides that bind to the first extracellular domain of the Orai1 protein with high affinities and high specificities. These ligands were isolated from a random single-strand DNA (ssDNA) library with 40 randomized sequence positions, using synthesized peptides with amino acid sequences identical to the first extracellular domain of the Orai1 protein as the targets for SELEX selection. Seven aptamers were obtained after 12 rounds of SELEX. An enzyme-linked oligonucleotide assay (ELONA) was performed to determine the affinities of the aptamers. Aptamer Y1 had the highest affinity (Kd = 1.72×10-8 mol/L) and was selected for functional experiments in mast cells. Using LAD2 cells with the human high-affinity IgE receptor and Ca2+ release activation channel (CRAC), we demonstrated that Aptamer Y1 blocked IgE-mediated β-hexosaminidase release from cells triggered by biotin-IgE and streptavidin. A specific binding assay showed that Aptamer Y1 not only bound the Orai1 peptide specifically but also that the Orai1 peptide did not bind significantly to other random oligonucleotide molecules. Furthermore, Aptamer Y1 regulation of intracellular Ca2+ mobilization was investigated by probing intracellular Ca2+ with a Fluo-4-AM fluorescent probe. We found that Aptamer Y1 inhibits Ca2+ influx into antigen-activated mast cells. These results indicate that the target of Aptamer Y1 in the degranulation pathway is upstream of Ca2+ influx. Therefore, these oligonucleotide agents represent a novel class of CRAC inhibitors that may be useful in the fight against allergic diseases.
authors.affiliation: Department of Dermatology, Institute of Battle Surgery, Daping Hospital, Third Military Medical University, Chongqing, 400042, China.
authors.forename: Renshan
authors.initials: R
authors.lastname: Sun
authors.affiliation: Department of Dermatology, Institute of Battle Surgery, Daping Hospital, Third Military Medical University, Chongqing, 400042, China.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Institute of Combined Injury, College of Military Preventive Medicine, Third Military Medical University, Chongqing, 400038, China.
authors.forename: Xinze
authors.initials: X
authors.lastname: Ran
authors.affiliation: Department of Dermatology, Institute of Battle Surgery, Daping Hospital, Third Military Medical University, Chongqing, 400042, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics; D004798:Enzymes / Q000737:chemistry; D006801:Humans; D006967:Hypersensitivity; D007073:Immunoglobulin E / Q000737:chemistry; D008024:Ligands; D008407:Mast Cells / Q000378:metabolism*; D018613:Microscopy, Confocal; D000071740:ORAI1 Protein / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D052156:SELEX Aptamer Technique
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D004798:Enzymes; D008024:Ligands; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D009841:Oligonucleotides; D007073:Immunoglobulin E; D002118:Calcium