Interaction of chemically modified antisense oligonucleotides with sense DNA: A label-free interaction study with reflectometric interference spectroscopy

Antisense oligonucleotides (ON) are regarded as potential therapeutic agent s for controlling gene expression at the mRNA level. The strength of the in teraction with the target sequence is one critical factor for the therapeut ic efficiency of an ON. Herein, the results of studies on antisense 15mer a nd 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomeno n of multidrug resistance. The effects of backbone modification (DNA, phosp horothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocophe rol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalig nments (1 to 3 bases) on interaction kinetics and binding strength were inv estigated. The interaction of an immobilized sense strand with the dissolve d antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS), Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/-0.2) x 10(4) M-1 s(-1) (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/-0.1) x 10(4) M -1 s(-1) (hexadecyl-modified strand). Binding constants varied from 1.9 (+/ - 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/-0.4) x 10(7) M-1 (to copherol modification). Phosphorothioate ON showed a reduction in binding s trength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.