DETECTION AND KINETIC-STUDIES OF TRIPLEX FORMATION BY OLIGODEOXYNUCLEOTIDES USING REAL-TIME BIOMOLECULAR INTERACTION ANALYSIS (BIA)

Real-time biomolecular interaction analysis (BIA) has been applied to tripler formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand, Sequence-specific tripler formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by tripler recognition, The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed, A preliminary kinetic analysis of tripler formation at pH 5.2 by an 11 mer third strand containing thymine, cytosine and uracil is reported, Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M(-1) S-1 and (8.1 +/- 1.9) x 10(-5) s(-1), respectively.