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Polyamines are polycationic molecules that bind to negatively charged cellular constituents such as nucleic acids and proteins. The naturally occurring polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), are present in millimolar levels in living cells and play important roles in growth and proliferation. Although polyamines perform vital functions in the cell, the molecular basis of the interaction of polyamines with biomolecules is not fully understood. Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent associations involving biomolecules. Using ESI-ion trap mass spectrometry, we have investigated the binding of polyamines to duplex oligonucleotides. Centrifugal filtration was used to desalt the oligodeoxyribonucleotides, 5'-CCG CGC GCG CC-3' (CC), 5'-GGC GCG CGC GG-3' (GG), 5'-GCG CAA TTG CGC-3' (dodecamer) and 5'-GGG GAA TTC CCC-3' (consensus). The complementary strands GG and CC were annealed to give the "heteroduplex". The self-complementary sequences were also annealed. Final solutions of 7 μM duplex concentration were used. Noncovalent complexes of polyamines with duplex oligonucleotides were observed by ESI-MS at micromolar polyamine concentrations suggesting that such interactions may occur in-vivo. Multiple adducts of polyamines were formed with the duplex oligonucleotides. Full scan and MS/MS spectra of the noncovalent complexes were obtained. Dissociation of the polyamine and loss of bases were the major pathways during MS/MS of 1:1 polyamine-duplex adducts. Higher order complexes (e.g., 2:1, 3:1) dissociated mainly with loss of the polyamine molecule. Dissociation curves obtained from MS/MS spectra of adducts revealed that the half-maximal percent relative collision energy required to dissociate the polyamines from the complex were in the order SPM > SPD > PUT. Dissociation constants for polyamine-duplex binding were determined from binding curves. Spermidine competed effectively with the intercalator ethidium for heteroduplex binding. On the basis of the half-maximal energy and the dissociation constants, the polyamines had a higher affinity for the AT containing sequences as compared to the only GC containing sequences. These results suggest a base or sequence selectivity in polyamine binding. Although electrostatic effects may play a dominant role in the binding of polyamines to duplex oligonucleotides, our results suggest that secondary base specific effects may also be present.