Solution Structure of a DNA Complex with the Fluorescent Bis-Intercalator TOTO Determined by NMR Spectroscopy

Abstract

We have used two-dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with the bis-intercalating dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-(3-methyl -2,3- dihydrobenzo-1,3-thiazolyl-2-methylidene)qui nolinium] tetraiodide (TOTO). The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the TOTO complex from both A-form and B-form dsDNA starting structures. The root-mean-square (rms) deviation of the coordinates for the 40 structures of the complex was 1.45 A. The local DNA structure is distorted in the complex. The helix is unwound by 60 degrees and has an overall helical repeat of 12 base pairs, caused by bis-intercalation of TOTO. The poly(propylenamine) linker chain is located in the minor groove of dsDNA. Calculations indicate that the benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTO molecule. The site selectivity of TOTO for the CTAG-CTAG site is explained by its ability to adapt to the base pair propeller twist of dsDNA to optimize stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. There is a 3000-fold fluorescence enhancement upon binding of TOTO to dsDNA. Rotation about the cyanine methine bonds is possible in free TOTO, allowing relaxation nonradiatively. When bound to dsDNA, the benzothiazole ring and the quinolinium ring are clamped by the nucleobases preventing this rotation, and the chromophore loses excitation energy by fluorescence instead.

The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules

“…These dimers consist of two identical fluorophores linked by a short flexible chain (EBEB (Ϸ1.54 nm), TOTO and YOYO (Ϸ1.68 nm)). The linker allows both dye groups to intercalate simultaneously in close proximity, generally 2 bp apart (16). It is consistent with our theory that such molecules should interact causing a significant decrease in polarization.…”

“…At an incorporation density of 1:10, TO and YO will have on average two neighbors within energy transfer range in each direction. These molecules are held in a parallel orientation between the steps of the DNA ladder (16), further increasing the chance of dipole interactions. In contrast, at an incorporation density of 1:100, the average distance between dye molecules is 340 Å; well beyond the range of energy transfer for all dyes studied.…”

“…For our purposes, we estimate P 0 from Figure 1 to be 0.45 for EB and TO and 0.48 for YO. This model assumes that the intercalators are inserted perpendicular to the helical axis of the DNA (16), and that insertion of a dye molecule unwinds the DNA helix by 20°, and expands it by 3.4 Å. These two parameters, along with the Förster's radius are the only variables used in the modeling.…”

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

“…generate forces as high as ∼60 pN. Also, structural studies of the DNA-YOYO-1 and DNA-TOTO complexes shows that this structure is more consistent with B than A form DNA (A form is 33% compressed compared with B), thus possibly accounting for comparable packaging inhibitory effects of 9AA and the more strongly binding YOYO-1 in our experiments (21,22). Overall, our direct FRET measurements showing likely DNA compression in the stalled Y-DNA stem and these results showing ID removal during translocation by the powerful motor appear to be consistent and confirmatory (8,10).…”

Structure–Fluorescence Contrast Relationship in Cyanine DNA Intercalators: Toward Rational Dye Design

“…The suppression of the excitonic interaction is due not so much to the distance between the two chromophores in DNA as to their quasi-orthogonal relative orientation (NMR measurements of TOTO-1 bound to DNA indicate a dihedral angle 838 between the chromophoric units). [53] Indeed, it was demonstrated that the interaction between the chromophoric units is sufficient to allow high enough excitation energy to hop between the two chromophores within a few picoseconds. [39,41,54] Table 1.…”