Affinity of aptamers binding 33-mer gliadin peptide and gluten proteins: Influence of immobilization and labeling tags.
Aptamers are starting to increase the reagents tool box to develop more sensitive and reliable methods for food allergens. In most of these assays, aptamers have to be modified for detection and/or immobilization purposes. To take full advantage of their affinity, which decisively influence the detectability, these modifications must be faced rationally. In this work, a recently developed aptamer for an immunotoxic peptide of gliadin associated to celiac disease is used in different configurations and modified with various markers and anchored groups to evaluate the influence of such modifications on the real affinity. The interaction in solution with the peptide is strong for a relatively small molecule (Kd = 45 ± 10 nM, 17 °C) and slightly stronger than that for the immobilized intact protein due to a cooperative binding effect. Comparatively, while only minor differences were found when the peptide or the aptamer were immobilized, labeling with a biotin resulted preferable over fluorescein (Kd = 102 ± 11 vs 208 ± 54 nM, 25 °C). These findings are of prime importance for the design of an aptamer-based analytical method for gluten quantification.
Dpto. Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: s.amaya_g@yahoo.es.
Sonia
S
Amaya-González
Dpto. Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: UO194986@uniovi.es.
Laura
L
López-López
Dpto. Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: mirandarebeca@uniovi.es.
Rebeca
R
Miranda-Castro
Dpto. Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: santosnoemi@uniovi.es.
Noemí
N
de-los-Santos-Álvarez
Dpto. Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: amir@uniovi.es.
Arturo José
AJ
Miranda-Ordieres
Dpto. Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: mjlc@uniovi.es.
María Jesús
MJ
Lobo-Castañón
D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D002446:Celiac Disease / Q000175:diagnosis; D058266:Dielectric Spectroscopy; D005903:Gliadin / Q000032:analysis*; D005983:Glutens / Q000032:analysis*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020349:Surface Plasmon Resonance
D052157:Aptamers, Nucleotide; D005983:Glutens; D005903:Gliadin
33-mer Peptide; Aptamer; Binding constant; Gliadin; Gluten