Hydration of the double helix plays an important role in the conformational transition from right handed B-DNA to left handed Z-DNA, which may be a switch in regulating genetic expression. The Z-DNA conformer is more sparsely hydrated and has a larger solvent exposure of the nonpolar base-pair surfaces. We tested the transitional effects of multiple osmolytes with varying degrees of hydrophobicity.

1. Introduction
Hydration of the double helix plays an important role in the transition
from right handed B-DNA to left handed Z-DNA, which may be a switch
in regulating genetic expression. The Z-DNA conformer is more
sparsely hydrated and has a larger solvent exposure of the nonpolar
base-pair surfaces (Rich et al., 1984). In previous studies applying an
osmotic stress by adding a neutral osmolyte shifted the B-Z equilibrium
toward Z-DNA and decreased the required concentration of transition
metal complexes required to achieve the transition (Preisler et al.,
1995, 2007, 2009; Ashman et al., 2011).
We hypothesized that more hydrophobic hydroxylic molecules would
interact more favorably with the exposed nonpolar surfaces in Z-DNA,
resulting in a weaker exclusion. The less hydrophobic osmolytes would
be more strongly excluded and would favor formation of the more
extensively hydrated B-DNA conformer.
.
Methods
Poly[d(G-C)] was dialyzed against a buffer to remove contaminating
polyvalent cations. The stock DNA concentration was determined by
UV absorbance using a Cary 100 spectrophotometer. Transition metal
coordination compounds were synthesized and purified using published
procedures. Alcohols, polyols and other osmolytes were purchased to
99% or higher purity from Sigma-Aldrich and were used without further
purification. Osmolyte concentrations in samples for circular dichroism
(CD) were determined gravimetrically. CD measurements were
performed at 25°C in a Pelletier-controlled Jasco J-815
spectropolarimeter.
Results & Discussion
Figure 1 shows CD spectra for solutions containing 40 µM poly[d(G-
C)] and 1 molal methanol with increasing concentrations of the +3
complex cobalthexammine. At 0 and 2 µM cobalthexammine the DNA
was in the B-conformation, as indicated by the negative band at 253 nm
and positive band at 280 nm. As the concentration of complex was
increased, the conformation shifted to Z-DNA, with the appearance of a
negative band at 293 nm and a positive band at 270 nm (Rich et al.,
1984). The transition midpoint was observed at about 4 µM
cobalthexammine, compared to 5 µM in the absence of osmolyte (see
Figure 2). These results are consistent with a shift in the equilibrium
towards Z-DNA produced by osmotic stress.
Figures 3 and 4 show the equilibrium shift produced by methanol
and MPD in 40 µM poly[d(G-C)] solutions with increasing
concentrations of the +3 cobalthexammine and the +2
chlorocobaltpentammine complex, respectively.
Figure 5 compares spectra of 50 µM poly[d(G-C)] and 5 µM
cobalthexammine in solutions containing 20% (w/w) concentrations of
several structurally diverse osmolytes. The comparatively more
hydrophobic 2-propanol and glycerol shifted the equilibrium toward Z-
DNA, while the more polar urea and glycine betaine (urea has no alkyl
groups and a large number of polar bonds and betaine is charged)
produced less Z-DNA than the control with no osmolyte.
In future studies we plan to use X-ray scattering to allow a more direct
determination of the hydration of the double helix in osmolyte
solutions.
References
Ashman, I., Nguemeta, C.T., Ramos, S.L. and Preisler, R.S. (2011), Biophys. J.
Preisler, R. S., Chen, H. H., Colombo, M. F., Choe, Y., Short, B. J. and Rau, D. C. (1995)
Biochemistry 34, 14400-14407.
Preisler, R. S., Hurst, A., Suleyman, A. M., Wells, A. V. and Pribula, A. J. (2007),
Biophys. J. 92, 411a-412a.
Preisler, R. S., Ashman, A., Ha, B. and Pribula, A. J. (2009), Biophys. J. 96, 604a.
Rich, A., Nordheim, A. and Wang, A. H.-J. (1984) Ann. Rev. Biochem. 53, 791-846.
Stanley, C. and Rau, D.C. (2006), Biophys. J. 91, 912-920.
Acknowledgements
We thank Dr. Alan Pribula of Towson University for preparation of coordination
compounds and Dr. Donald C. Rau for useful discussions. This research was supported
by Undergraduate Research Grants (to C. Chaze) and by the Department of Chemistry,
Towson University.
.
Stanley and Rau (2006) expressed the relative hydrophobicities of
hydroxylated compounds in terms of the excess of alkyl carbons over
hydroxyl oxygens or Δ (C-O). We studied the osmotic stress produced
by the following solutes:
Figure 2 compares spectra of 40 µM poly[d(G-C)] in 1 molal solutions
of each of these osmolytes in the presence of 5 µM cobalthexammine.
Based on a comparison of ellipticities in the bands at around 250 and
290 nm, the relative extent of Z-DNA stabilization was as follows:
MPD ~ 1,4-butanediol > 2-butanol > 1-butanol ~ 2-propanol >
methanol > ethanol > no osmolyte.
The trend is consistent with our hypothesis, for the most part, with the
more hydrophobic compounds favoring Z-DNA more strongly.
Comparing the Effects of Different Osmolytes in the B-to-Z DNATransition
Charlotte A. Chaze and Richard S. Preisler
Department of Chemistry,Towson University,Towson MD
-8
-6
-4
-2
0
2
4
230 240 250 260 270 280 290 300 310 320
Ellipticity(mdeg)
Wavelength (nm)
Figure 1: B-Z Transition in 1 Molal Methanol Solutions
0 cohex
2 uM cohex
4 uM cohex
5 uM cohex
6 uM cohex
-3
-2
-1
0
1
2
3
230 240 250 260 270 280 290 300 310 320
Ellipticity(mdeg)
Wavelength (nm)
Figure 2: B-Z Transition in 5 uM Cobalthexammine Plus
Various Osmolytes
no osmolyte
methanol
ethanol
2-propanol
1-butanol
2-butanol
1,4-butanediol
MPD
-8
-6
-4
-2
0
2
4
230 240 250 260 270 280 290 300 310 320
Ellipticity(mdeg)
Wavelength (nm)
2 uM cohex
+ methanol
2 uM cohex
+ MPD
4 uM cohex
+ methanol
4 uM cohex
+ MPD
5 uM cohex
+ methanol
5 uM cohex
+ MPD
Figure 3: B-Z Transition in Cobalthexammine
and 1 Molal Osmolytes
-5
-3
-1
1
3
5
260 270 280 290 300 310 320
Ellipticity(mdeg)
Wavelength (nm)
Figure 4: B-Z Transition in
Cobaltchloropentammine and 1 Molal
Osmolytes
50 uM copent +
methanol
50 uM copent +
MPD
150 uM copent
+ methanol
150 uM copent
+ MPD
250 uM copent
+ methanol
250 uM copent
+ MPD
Compound Alkyl C Hydroxyl O Δ(C-O)
Methanol 1 1 0
Ethanol 2 1 1
2-propanol 3 1 2
1-butanol 4 1 3
2-butanol 4 1 3
1,4-butanediol 4 2 2
2-methyl-1,2-
Pentanediol
(MPD)
6 2 4
-8
-6
-4
-2
0
2
4
240 250 260 270 280 290 300 310 320
Ellipticity(mdeg)
Wavelength (nm)
Figure 5: B-Z Transition in 5 uM Cobalthexammine
Plus 20% Osmolyte
no osmolyte
PEG-1000
betaine
urea
2-propanol
glycerol
Structures of B-DNA (left)
and Z-DNA (right). B-DNA
is the physiologically
predominant form. The
base pairs in Z-DNA are
flipped 180° relative to
those in B-DNA.