Interaction of a photosensitizer methylene blue with various structural forms (cruciform, bulge duplex and hairpin) of designed DNA sequences.

Abstract

Functionally important, local structural transitions in DNA generate various alternative conformations. Cruciform is one of such alternative DNA structures, usually targeted in genomes by various proteins. Symmetry elements in sequence as inverted repeats are the key factor for cruciform formation, facilitated by the presence of the AT-rich regions. Here, we used biophysical and biochemical techniques such as Gel electrophoresis, Circular dichroism (CD), and UV-thermal melting analysis to explore the structural status of the designed DNA sequences, which had potential to form cruciform structures under physiological conditions. The gel electrophoresis analysis revealed that the designed 53-mer DNA oligonucleotide sequence CR forms an intermolecular bulge duplex with flanking ends, while another sequence CRC adopts an intramolecular hairpin structure with flanking ends. Their equimolar complex (CRCRC) bestowed much-retarded migration due to the formation of a quite intriguing cruciform structure. CD studies confirmed that all the alternative structures (cruciform, bulge duplex, and hairpin with flanking ends) exhibit characteristics of B-DNA type conformation. A triphasic UV-thermal melting curve displayed by the complex formed by the equimolar ratio (CRCRC) is also suggestive of the formation of the cruciform structure. The interaction studies of CR, CRC, and their equimolar complex (1:1) with a photosensitizer methylene blue (MB) indicated that MB could not stabilize the discrete structures formed by CR and CRC sequences, however, the cruciform structure showed a quite significant increment in the melting temperature. Such studies facilitate our understanding of various secondary structures possibly present inside the cell and their interactions with drug/dye molecules.