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 agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved 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 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.
Sauer
M
M
Institute for Physical and Theoretical Chemistry, University of Tübingen, Germany.
Brecht
A
A
Charissé
K
K
Maier
M
M
Gerster
M
M
Stemmler
I
I
Gauglitz
G
G
Bayer
E
E
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods*; D013816:Thermodynamics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016376:Oligonucleotides, Antisense; D004247:DNA