Nucleic Acids Research, Vol 25, Issue 10 1890-1896, Copyright © 1997 by Oxford University Press
AA Moraru-Allen, S Cassidy, JL Asensio Alvarez, KR Fox, T Brown and AN Lane
Intercalating ligands may improve both the stability and sequence
specificity of triple helices. Numerous intercalating drugs have been
described, including coralyne, which preferentially binds triple helices,
though its sequence specificity has been reported to be low [Lee,J.S.,
Latimer,L.J.P. and Hampel,K.J. (1993) Biochemistry , 32, 5591-5597]. In
order to analyse the sequence preferences of coralyne we have used a
combination of DNase I footprinting, UV melting, UV-visible
spectrophotometry, circular dichroism and NMR spectroscopy to examine
defined intermolecular triplexes and intramolecular triplexes linked either
by hexaethylene glycol chains or by octandiol chains. DNase I footprinting
demonstrated that coralyne has a moderate preference for triplexes over
duplexes, but a substantial preference for TA.T triplets compared with CG.
C+triplets. The drug was found to have essentially no effect on the melting
temperatures of duplexes of the kind d(A)n.d(T)n or d(GA)n.d(TC)n. In
contrast, it increased the T m for triplexes of the kind d(T)nd(A)n.dTn,
but had little effect on the stability of d(TC)nd(GA).d(CT)n at either low
or high pH. On binding to DNA triplexes, there is a large change in the
absorption spectrum of coralyne and also a substantial fluorescence
quenching that can be attributed to intercalation. The changes in the
optical spectra have been used for direct titration with DNA. For triplexes
d(T)6d(A)6.d(T)6, the Kd at 298 K was 0.5-0.8 microM. In contrast, the
affinity for d(TC) nd(GA)n.d(CT)n triplexes was 6- to 10-fold lower and was
characterized by smaller changes in the absorption and CD spectra. This
indicates a preference for intercalation between TAT triples over
CG.C+/TA.T triples. NMR studies confirmed interaction by intercalation.
However, a single, secondary binding was observed at high concentrations of
ligand to the triplex d(AGAAGA-L-TCTTCT-L-TCTTCT), presumably owing to the
relatively low difference in affinity between the TA.T site and the
competing, neighbouring sites.
ARTICLES
Coralyne has a preference for intercalation between TA.T triples in intramolecular DNA triple helices
Department of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JJ, UK.
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