19th-banner-rev.gif

Mendel-Brno 2000

category image Volume: 17
Issue Number 6, Part 2
June 2000

DNA Intercalation in Solution and at the Electrode Surface

Electrochemical measurements with DNA-modified mercury electrodes have provided information about DNA structural changes, including formation of DNA single-strand breaks (sb) (1,2) and DNA conformational transitions induced by negative superhelix density (3). In these studies, a protocol involving DNA adsorption at the electrode surface followed by medium exchange(s) was used. This technique allowed to (i) significantly decrease the amount of DNA required for the analysis; (ii) remove low-molecular substances from the electrode during the washing step (which made it possible to study e.g., DNA interactions with electroactive substances) (iii) study interactions of surface-attached DNA with molecules in solution (1,2,4).

Here we used alternating current voltammetry at the mercury electrode to study conformational changes of DNA upon binding of DNA intercalators. Two tensammetric signals of double-stranded (ds) DNA, peak 2 [assigned to distorted or damaged dsDNA regions (4)] and peak 3 [related to a partial denaturation of dsDNA around ssb or molecule ends at the electrically charged mercury surface (1,4)] were sensitive to formation of DNA intercalative complexes in solution (prior to DNA adsorption at the electrode) (5). We assume that dsDNA (in absence of intercalators) adsorbed at the mercury surface predominantly by segments containing ssb or molecule ends (due to higher flexibility of the DNA molecule and random base unpairing at these sites), with intact dsDNA loops extending into the bulk of solution. Untwisting of DNA double helix by the intercalators resulted in an altered DNA adsorption. Unwound dsDNA segments attached to the electrode surface yielded increased peak 2. Due to higher overall affinity of underwound DNA to the surface, the preference for adsorption of nicked DNA segments and molecule ends might be reduced. After medium exchange and the intercalator removal, the surface-confined DNA probably adopted a restrained structure, with superhelical loops extending into the bulk of solution. Such a structure was probably resistant to DNA surface denaturation, which resulted in a decreased intensity of DNA peak 3. Similar effects were also observed when DNA was adsorbed at the electrode from a solution of low salt concentration. Upon formation of DNA ssb (both in solution prior to DNA adsorption, and in surface-confined DNA), the specific AC voltammetric behavior of DNA-intercalator complexes was eliminated.

This work was supported by grants of the Grant Agency of the Academy of Sciences of the Czech Republic No. A4004801, and of the Grant Agency of the Czech Republic No. 204/98/P091.

References

1. M. Fojta and E. Palecek, Anal. Chim. Acta 342, 1-12 (1997).
2. M. Fojta, T. Kubicarova and E. Palecek, Electroanalysis 11, 1005-1012 (1999).
3. M. Fojta, R. P. Bowater, V. Stankova, L. Havran, D. M. J. Lilley and E. Palecek, Biochemistry 37, 4853-4862 (1998).
4. E. Palecek, Electroanalysis 8, 7 - 14 (1996).
5. M. Fojta, L. Havran, T. Kubicarova and J. Fulneckova, Electroanalysis in press(2000).

Miroslav Fojta, Ludek Havran, Lucie Kovarova, Jana Fulneckova and Tatiana Kubicarova

Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno
fojta@ibp.cz

$15.00