THERMODYNAMIC, KINETIC, AND CONFORMATIONAL PROPERTIES OF A PARALLEL INTERMOLECULAR DNA TRIPLEX CONTAINING 5' AND 3' JUNCTIONS

The interaction of the 11-mer oligodeoxypyrimidine d(TCTTCTUTCCT) with the 17 bp duplex d(CCGTAGAAGAAAGGACG).d(CGTCCUTTCTTCTAGCG) in forming an intermolecular DNA tripler has been examined in solution by surfac e plasmon resonance (SPR), UV thermal denaturation, circular dichroism (CD), and NMR methods. Thermodynamic data were also acquired for the shorter 15 bp target duplex d(CGCTAGAAGAAAGGA).d(TCCUTTCTTCTAGCG), whi ch forms a 3' flush-ended parallel tripler. CD titrations at pH 5 gave a tripler --> (duplex + strand) dissociation constant K-d Of 0.5 mu M at 15 degrees C and similar to 2 mu M at 25 degrees C for both the 11 -15.15 and 11-17.17 systems, in agreement with analysis of the UV melt ing data and a direct calorimetric measurement. In contrast, the ''app arent'' K-d value determined by SPR was 10-20-fold smaller. The rate c onstant for dissociation (kd) of the third strand from the tripler was found to be similar to 0.0002 s(-1) at 25 degrees C by SPR. The rate constant for exchange between the tripler and duplex states determined by NMR was similar to 2 s(-1) at 40 degrees C. The dissociation kinet ics measured by SPR are considerably underestimated, which largely acc ounts for the poor estimation of K-d using this technique. Extensive H -1 NMR assignments were obtained for both the 17 bp DNA duplex and the tripler. Large changes in chemical shifts were observed in the purine strand of the host duplex, but only small shift changes were induced in the complementary pyrimidine strand. Dramatic differences in shifts were observed for the G and A residues, especially in the minor groov e, consistent with only small, localized conformational changes in the underlying duplex. The magnitude of the shift changes decreased to ba seline within one base of the 3' tripler-duplex junction and over two to three bases at the 5' junction. Chemical shift changes at the 5' ju nction suggest small conformational anomalies at this site. COSY and N OESY spectra indicate that the nucleotides are in the ''S'' domain in both the tripler and duplex states. These data rule out major conforma tion changes at the tripler-duplex boundaries. NOEs between pyrimidine s in the third strand and those in the duplex showed proximity for the se bases in the major groove, which could be ascribed to buckling of t he Hoogsteen bases out of the plane of the Watson-Crick base pairs.