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

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 degrees C and 37 degrees 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) x 10(5) M-1 for HSA at 37 degrees C and 2.47 (+/- 0.01) x 10(5) M-1 for HSA at 25 degrees C), while the high molecular weight aptamer quenched albumins by static process (estimated Stern-Volmer constant: 4.05 (+/- 0.01) x 10(5) M-1 for HSA at 37 degrees C and 6.20 (+/- 0.01) x 10(5) M-1 for HSA at 25 degrees 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 degrees C. Similarly, although short aptamer does 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 R-1/R-2 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. (C) 2013 Elsevier B.V. All rights reserved.