Studies on the base pair binding specificity of CC-1065 to oligomer duplexes

In this paper, we report on the base pair binding specificity of CC-1065 to oligomer duplexes of several lengths and base composition as determined by circular dichroism (CD) methods. The oligomers are synthesized using the phosphoramidite triester coupling approach and purified by either polyacrylamide gel electrophoresis or anion-exchange HPLC. CC-1065 binds by two different mechanisms to form a reversibly bound species and an irreversibly bound species, both of which show intense induced CD bands. The reversible to irreversible binding transition is characterized by a shift of the CD band to shorter wavelength (392→371 nm) characteristic of the reaction between the cyclopropyl group of CC-1065 and the N-3 of adenine. The induced CD acquired by the CC-1065 chromophore increases with increasing oligomer chain length and with an increase in the number of bases to the 5′ end of the site of attachment whether a purine or a pyrimidine is at position 5 (or 4) from the site of attachment at the 3′ end is not crucial for binding. The binding sequences 5′-GATAT and 5′-GTATA show a slower conversion to an irreversibly bound species relative to the preferred sequences 5′-AAA and 5′-TTA. A G base pair at position 3 in 5′-AAGAA hinders the formation of the irreversibly bound species relative to the 5′-GAAAA and 5′-AGAAA sequences. Very stable reversible binding is observed with the sequences 5′-GAATT and 5′-AAGAA. The sequences 5′-GCGAA and 5′-AGAG also show reversible binding but are characterized by a relatively small induced molar ellipticity which decreases with time. These binding characteristics signify weaker binding for these sequences. Overall, these binding studies agree with earlier sequence studies which found two preferred binding sequences, 5′-AAAAA and 5′-PuNTTA, with CC-1065 attached to the 3′ end of the binding site. Furthermore, according to studies of the oligomer 5′-CGCGAATTCGCG-3′ under different experimental conditions, the annealing conditions of this work produced oligomer duplex structures, not hairpin structures. In these studies, we found that CC-1065 binds very little or not at all to hairpin structures.