Calcium Channel-specific Oligonucleotide Aptamers Selected By SELEX And Its Effects On Mast Cell Activation

Objective and methodsCalcium influx is necessary and sufficient for mast cells activation. Calciumrelease-activated calcium (CRAC) channels mediate calcium influx in mast cells. The twoproteins which constitute CRAC functions is Orai1and stromal interaction molecule1(STIM1). To block calcium influx in mast cells may inhibit mast cell activation and thus forallergy treatment. Systematic evolution of ligand by exponential enrichment(SELEX) is aprocess for isolating DNA or RNA sequences with high affinity and selectivity formolecular targets from random sequence libraries. These sequences are commonly referredto as aptamers. Aptamers have shown extensively and potential applications in clinicalpractice. In this study, we selected aptamers to the first extracellular region of Orai1proteinby SELEX technique. Then using LAD2mast cell line degranulation model mediated byIgE, we studied the inhibitory effect of aptamers on calcium influx of mast cells. Thepossibility of aptamers which inhibit mast cell activation was explored by inhibitingcalcium influx. The study is divided into three parts:(1) LAD2human mast cells would becultured for establishing a model of mast cell activation mediated by IgE.(2) we confirmedthat the first extracellular region has Orai1protein specificity by protein sequencealignment. The oligonucleotide aptamers would be selected targeting the first extracellularregion by SELEX technology with enzyme-linked polystyrene plate as medium.(3) Thechanges of intracellular calcium concentration in LAD2cells would be observed by laserconfocal microscopy. Using mast cell degranulation model mediated by IgE. The inhibitoryeffects on mast cell activation would be tested.Results:1. LAD2human mast cells were cultured and LAD2degranulation model wasestablished. Doubling time of LAD2cell was10days. Cells staining and electronmicroscopy showed perinuclear particles of varying sizes. In the culture system of1×106cells/mL, we optimized the best concentration of biotin-IgE sensitization500ng/mL and, streptavidin activating LAD2cells1000ng/mL. The histamine and β-hexosaminidaserelease reached the best degree.2. SELEX screening required12cycles of enrichment in this study. The affinity ofenrichment sequence library increased gradually with increased cycles, the affinity reachedthe highest value in the eleventh cycle. We obtained seven aptamers from the eleventhenrichment library. AptamerY1showed the highest affinity. The specificity of theAptmerY1binding to Oari1was proved by ELISA. The affinity constant of AptamerY1(Kd value) was1.72×10-8mol/L.3. AptamerY1obtained from the part2decreased the calcium entry and inhibited theβ-hexosaminidase release from LAD2cells by95%, it was obseraved by laser scanningconfocal microscopy. It is possible that AptamerY1inhibition effects on mast celldegranulation was achieved by inhibiting calcium influx.Conclusion:Human mast cells line LAD2degranulation model mediated by IgE was established.The seven oligonucleotide aptamers targeting the first extracellular region of Orai1proteinwere obtained by SELEX technology. The AptamersY1can decrease effectively calciumentry and inhibit effectively the release of β-hexosaminidase from LAD2cells. Mast cellactivation can be inhibited through CRAC channel. The study may provide a new way forallergy therapy by inhibiting calcium influx and mast cell activation.