A fluorescent spectroscopy and modelling analysis of anti-heparanase aptamers–serum protein interactions

•A novel approach to study drug-protein interactions is presented, for two anti-heparanase aptamers.•The short aptamer binds collisionally, whereas the long one forms complexes with serum albumins.•Quenching constants for the interactions of each aptamer with serum albumins were calculated.•Binding sites, and the way aptamers could be docked to serum proteins was studied by modelling.•The most promising aptamer for clinical use is the short one due to its higher specificity for heparanase.

Aptamers are short, single stranded oligonucleotide or peptide molecules that bind a specific target molecule and can be used for the delivery of therapeutic agents and/or for imaging and clinical diagnosis. Several works have been developed aiming at the production of aptamers and the study of their applications, but few results have been reported on plasmatic dynamics of such products. Aptamers against the heparanase enzyme have been previously described. In this work, the interactions of two constructs of the most promising anti-heparanase aptamer (molecular weights about 9200 Da and 22000 Da) to human and bovine serum albumins were studied by fluorescence quenching technique. Stern–Volmer graphs were plotted and quenching constants were estimated. Stern–Volmer plots obtained from experiments carried out at 25 °C and 37 °C showed that the quenching of fluorescence of HSA and BSA by the low molecular weight aptamer was a collisional phenomenon (estimated Stern–Volmer constant: 3.22 (±0.01) × 105 M−1 for HSA at 37 °C and 2.47 (±0.01) × 105 M−1 for HSA at 25 °C), while the high molecular weight aptamer quenched albumins by static process (estimated Stern–Volmer constant: 4.05 (±0.01) × 105 M−1 for HSA at 37 °C and 6.20 (±0.01) × 105 M−1 for HSA at 25 °C), interacting with those proteins constituting complexes. Linear Stern–Volmer plot from HSA titrated with the low MW aptamer suggested the existence of a single binding site for the quencher in this albumin. Differently, for aptamer 2, the slightly downward curvature of the Stern–Volmer plot of the titration for that albumin suggested a possible conformational change that led to the exposition of lower affinity binding sites in HSA at 25 °C. Similarly, although short aptamerdoes not appear to form a stable complex (collisional interaction), the longer aptamer is found to form a stable complex with HSA. In addition, the behaviour of quenching curves for HSA and BSA and values estimated for ratio R1/R2 from model developed by Silva et al. suggest that the primary binding site in both aptamers is located closer to the tryptophan residue in sub domain IIA. It is likely that both aptamers are competing for the same primary site in albumin.

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