Duplex-selective ruthenium-based DNA intercalators

Abstract

We report the design and synthesis of small molecules that exhibit enhanced luminescence in the presence of duplex rather than single-stranded DNA. The local environment presented by a well-known [Ru(dipyrido[3,2-a:2',3'-c]phenazine)L2 ](2+) -based DNA intercalator was modified by functionalizing the bipyridine ligands with esters and carboxylic acids. By systematically varying the number and charge of the pendant groups, it was determined that decreasing the electrostatic interaction between the intercalator and the anionic DNA backbone reduced single-strand interactions and translated to better duplex specificity. In studying this class of complexes, a single Ru(II) complex emerged that selectively luminesces in the presence of duplex DNA with little to no background from interacting with single-stranded DNA. This complex shows promise as a new dye capable of selectively staining double- versus single-stranded DNA in gel electrophoresis, which cannot be done with conventional SYBR dyes.

Keywords: DNA; bioinorganic chemistry; gels; luminescence; ruthenium.

Figure 1

Schematic representation of intercalator complexes synthesized for this study. Increasing contributions from both steric and electrostatic contributions were systematically studied through synthetic changes in the ancillary ligand side chains. X = PF₆⁻ as synthesized or Cl⁻ after salt exchange.

Figure 2

Characteristic luminescence intensities of complexes at 3 µM with no or one pendant chain upon interaction with 10 µM of ssDNA and dsDNA in PBS (10 mM, pH 7.5). a) Emission profiles of complex 1 in the presence of ssDNA vs dsDNA and for b) complexes 2 and3, respectively. The temperature of the samples was maintained at 25 °C, and the counter anion for all complexes was Cl⁻. c) The 18 base DNA sequences used in these fluorescence assays.

Figure 3

Characteristic fluorescence intensities of complexes at 3 µM with two pendant chains upon interaction with 10 µM of dsDNA and ssDNA in PBS (10 mM, pH 7.5) at 25 °C. The counter anion for all complexes was Cl⁻.

Figure 4

Characteristic fluorescence intensities of complexes at 3 µM with three pendant chains upon interaction with 10 µM dsDNA and ssDNA in PBS (10 mM, pH 7.5) at 25 °C. The counter anion for all complexes was Cl⁻.

Figure 5

Gel electrophoresis results showing the relative intercalative nature of each Ru complex when run in a mixture of ssDNA versus dsDNA samples. Samples included (from left to right) are complex 10, complex6, complex 3, complex 1, and complex 7. Degree of intercalation is indicated by the relative fluorescence intensities of the individual gel bands. Apparent from the differential staining of the DNA in each lane is that the neutral Ru^II complex can distinguish ssDNA from dsDNA, whereas the more positively charged Ru complexes cannot. Electrostatic repulsion between the negatively charged Ru complex and the negatively charged DNA backbone may significantly hinder intercalation, thus resulting in little to no fluorescence even in the presence of dsDNA. The gel to the right shows single and double stranded DNA samples post staining with the commercially available 1× SYBR Gold stain (Invitrogen) for comparison.