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.

Based on the application of cationic polystyrene nanoparticles, a novel method for solid-phase extraction of phosphorothioate oligonucleotides from human plasma has been developed. A high binding affinity, which is required for an effective isolation out of complex mixtures, is mediated by hydrophobic and multiple electrostatic interactions between the oligonucleotides and the nanoparticles. The principle of the method is based on a pH-controlled adsorption/desorption mechanism. Analysis of the extracted samples was performed by capillary gel electrophoresis. Extraction conditions were optimized, providing the isolation of oligonucleotides (> or = 10 nucleotide units) in high yields and purity even at concentrations in the low-nanomolar range (down to 5 nM). The low salt contamination of the samples allows their direct analysis by electrospray mass spectrometry. The combined linearity and accuracy of the assay together with absolute recovery rates in the range of 60-90% indicate that the developed solid-phase extraction method is generally applicable to quantitation of oligonucleotides in human plasma. Further improvement was achieved with an optimized carrier system of 2-fold enlarged particles which reduces the time consumption of the extraction procedure to approximately 30 min.

The collision induced dissociations of oligodeoxynucleotides, 5' and 3'-terminal modified oligodeoxynucleotides and oligophosphorothioates, have been studied by using pneumatically assisted electrospray ionization mass spectrometry on a triple quadrupole instrument. Fragment ions were either produced in the collision gas cell of the triple quadrupole mass spectrometer or by nozzle-skimmer fragmentation. Sequence information was obtained for oligomers up to 21 bases and for 5'-and 3'-terminal modified 15-mers. Main fragments observed for oligodeoxynucleotides resulted from a-B-and w-type cleavages, and were most prominent when the lost base was guanine or cytosine. Positions containing thymine exhibited a low signal intensity for the corresponding a-B-and w-type fragment ions. Internal fragment ions containing from one up to four bases resulted from two subsequent a-b-and w-type cleavages, and were most prominent when both bases lost were guanine and/or cytosine. resulting from a-B-, w-, b-, c-, x-and y-type cleavages were observed. In contrast to oligodeoxynucleotides none of the fragmentation reactions seems to be favoured, resulting in more sequence specific fragment ions. Compared to oligodeoxynucleotides, internal fragment ions were less prominent for oligophosphorothioates, possibly due to the many low energy fragmentation reactions possible for the intact oligomer and each fragment ion. For oligophosphorothioates fragments

No abstract

The collision induced dissociations of oligodeoxynucleotides, 5' and 3'-terminal modified oligodeoxynucleotides and oligophosphorothioates, have been studied by using pneumatically assisted electrospray ionization mass spectrometry on a triple quadrupole instrument. Fragment ions were either produced in the collision gas cell of the triple quadrupole mass spectrometer or by nozzle-skimmer fragmentation. Sequence information was obtained for oligomers up to 21 bases and for 5'-and 3'-terminal modified 15-mers. Main fragments observed for oligodeoxynucleotides resulted from a-B-and w-type cleavages, and were most prominent when the lost base was guanine or cytosine. Positions containing thymine exhibited a low signal intensity for the corresponding a-B-and w-type fragment ions. Internal fragment ions containing from one up to four bases resulted from two subsequent a-b-and w-type cleavages, and were most prominent when both bases lost were guanine and/or cytosine.For oligophosphorothioates fragments resulting from a-B-, w-, b-, c-, x-and y-type cleavages were observed. In contrast to oligodeoxynucleotides none of the fragmentation reactions seems to be favoured, resulting in more sequence specific fragment ions. Compared to oligodeoxynucleotides, internal fragment ions were less prominent for oligophosphorothioates, possibly due to the many low energy fragmentation reactions possible for the intact oligomer and each fragment ion. # 1998 John Wiley & Sons, Ltd. Received 29 August 1997; Revised 2 February 1998; Accepted 5 February 1998 Rapid Commun. Mass Spectrom. 12, 389-397 (1998 The antisense strategy, a promising new pharmaceutical therapy, uses synthetic oligodeoxynucleotides or analogous structures to bind messenger-ribonucleic acid (m-RNA) thereby inhibiting the synthesis of specific proteins.