Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair

Abstract

Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins.

Figure 1.

DNA sequences investigated. Position of donor, tC^O, in red and acceptor, tC_nitro, in green. Cytosines marked by a star are methylated unless they are replaced by tC_nitro. In the hemimethylated sequence the donor strand containing tC^O is not methylated. The DX and AY notation of sequences reflect the position of the donor, D, and the acceptor, A, in the sequence. The red box marks expected position of the extruded base pair according to literature (16,17).

Figure 2.

Top and side view of the B- and Z-DNA structures showing the positions and orientations of tC^O (red) and tC_nitro (green) for different base-pair separations. Arrows in the top view indicate the direction of the transition dipole moments of the tC^O/tC_nitro chromophores. In the top view, transparency increases with the number of base pairs separating tC^O and tC_nitro.

Figure 3.

CD spectra and FRET efficiencies calculated from time-resolved emission measurements of the (GC)₇ hairpin sequence at different salt concentrations. CD spectra of modified (GC)₇ hairpins at (A) 100 mM, (B) 3 M and (C) 4.5 M NaCl, the reference hairpin being the corresponding unmodified sequence with only natural nucleobases. To facilitate comparison, the CD spectra of the reference structures at the three salt conditions are displayed together in panel (D). (E) FRET efficiencies calculated from time-resolved measurements of the (GC)₇ hairpin structures at the three salt conditions. Measurements performed at room temperature in pH 7.5 buffer (25 mM PBS).

Figure 4.

CD spectra and time-resolved FRET efficiencies for the methylated and hemimethylated B-Z DNA junction sequences at low and high salt concentrations. CD spectra of the methylated B-Z DNA junction sequences (MeBZ) in (A) 100 mM and (B) 4.5 M NaCl. (C) CD spectra of the hemimethylated B-Z DNA junction sequences (hMeBZ) at 4.5 M NaCl. (D) CD spectra of the reference methylated (MeBZ), hemimethylated (hMeBZ) and unmethylated (BZ) B-Z DNA junction sequences at 100 mM and 4.5 M NaCl. (E) Time-resolved FRET efficiencies of the methylated (patterned bars) and hemimethylated duplexes at 100 mM and 4.5 M NaCl. The reference sample in panels A to C is the sequence containing no tricyclic cytosine molecules. Measurements performed at room temperature in pH 7.5 buffer (25 mM PBS).

Figure 5.

Kinetics of B- to Z-DNA transition of (GC)₇ hairpin structure. Measurements were performed at 4.5 M NaCl (left) and 3 M NaCl (right) and 20°C by monitoring the fluorescence intensity at 460 nm (data points). Lines are mono-exponential fits of the experimental data.