An. Veselkov et al., H-1-NMR STUDY OF COMPLEX-FORMATION BETWEEN ETHIDIUM-BROMIDE AND PALINDROMIC SEQUENCE 5'-D(GPCPGPAPAPGPC) IN AQUEOUS-SOLUTION, Molecular biology, 32(4), 1998, pp. 527-535
Complex formation between the phenanthridine dye ethidium bromide and
a palindromic heptadeoxynucleotide sequence 5'-d(GpCpGpApApGpC), which
can form a hairpin, in water-salt solution was studied by one-dimensi
onal H-1, and two-dimensional H-1 and P-31 NMR spectroscopy. Two-dimen
sional homonuclear H-1 NMR spectroscopy (2D TOCSY and 2D NOESY) and he
teronuclear H-1-P-31 NMR spectroscopy were used to completely assign t
he signals of protons and phosphorus nuclei, and to examine the ethidi
um bromide interaction with heptanucleotide. The concentration (at 298
and 308 K) and temperature (in the range 278-353 K) dependences of th
e chemical shifts of protons were obtained. Various complexation schem
es taking into account different molecular associates in solution were
analyzed. The equilibrium constants and the limit chemical shifts of
ethidium bromide protons in complexes were determined. The portions of
complexes of different types (dye complexes with monomeric, hairpin,
and dimeric forms of heptanucleotide) in solution were found, and spec
ific features of the dynamic equilibrium were revealed as functions of
the heptanucleotide-to-dye concentration ratio and temperature. From
the calculated induced chemical shifts of ethidium bromide protons and
the two-dimensional NMR data, the most probable structures of interca
lated complexes with ethidium bromide bound to d(GC) sites of the hair
pin stem and dimer of heptanucleotide were constructed.