Synthesis of a liposome incorporated 1-carboxyalkylxanthine-phospholipid conjugate and its recognition by an RNA aptamer.
RNA or DNA aptamers have received much attention in recent literature as therapeutic agents and chromatographic matrices, however, their use in analytical methodologies is relatively unexplored. We describe here investigations aiming to combine this promising technology with versatile liposomes in a competitive assay format. Thus, a phospholipid derivative of an unsymmetrical 1,3-disubstituted xanthine (1-carboxyethyl-3-methylxanthine-DPPE) was prepared for incorporation into the lipid bilayers of dye-encapsulating liposomes. Its synthesis and characterization using GC-MS, (1)H NMR, and HPLC are described. Equilibrium filtration experiments using enzyme linked immunosorbent assays (ELISAs) were completed to assess the affinity for theophylline of an unmodified RNA aptamer and one that had been modified on the 3' end with biotin. A dissociation constant (K(d)) for theophylline with the unmodified RNA aptamer of 0.9muM and biotinylated aptamer of 1.0muM was determined which showed that this modification did not affect the aptamer's affinity using this technique. The observed K(d) values correlated well to the previously reported value of 0.6muM. Experiments were also carried out in a competitive manner with the prepared 1-carboxypropyl-3-methylxanthine intermediate, and the final 1-carboxypropyl-3-methylxanthine-DPPE conjugate once it had been incorporated into the bilayers of liposomes. The K(d) value for 1-carboxypropyl-3-methylxanthine was approximately 2.7muM. Finally, successful binding to theophylline-analog-tagged liposomes in a competitive assay format was shown versus liposomes prepared without the tag.
Edwards
Katie A
KA
Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, United States.
Baeumner
Antje J
AJ