Selective Interactions of a Few Acridinium Derivatives with Single Strand DNA: Study of Photophysical and DNA Binding Interactions Elizabeth Kuruvilla and Danaboyina Ramaiah* Photosciences and Photonics, Chemical Sciences and Technology DiVision, National Institute for Interdisciplinary Science and Technology, (Formerly, Regional Research Laboratory) (CSIR), TriVandrum 695 019, India ReceiVed: February 21, 2007; In Final Form: April 10, 2007 Novel acridinium derivatives 1-3, wherein steric factors have been varied systematically through substitution at the ninth position of the acridinium ring, were synthesized and their interactions with single strand and double strand DNA have been investigated through photophysical, biophysical, and microscopic techniques. The acridinium derivative 1 exhibited quantitative fluorescence yields (φ f = 1) and high lifetime of 35 ns, while significantly lower fluorescence yields of 0.11 and 0.02 and lifetimes of 3.5 and 1.2 ns were observed for 2 and 3, respectively. The derivatives 1 and 2 having 2-methylphenyl and 2,4-dimethylphenyl substituents at the ninth position of the acridinium ring showed selective interactions with single strand DNA (ssDNA) with association constants of K ssDNA ) 6.3-6.6 × 10 4 M -1 , while negligible interactions were observed with double strand DNA (dsDNA). In contrast, the derivative 3 with 2,6-dimethylphenyl substitution showed negligible interactions with both ssDNA and dsDNA. Studies with a series of 19-mer oligonucleotides indicate that these derivatives exhibit significant selectivity for the sequences rich in guanosine (ca. 3-fold) as compared to the cytosine-rich sequences. These derivatives with high water solubility and the ability to distinguish between ssDNA and dsDNA through changes in fluorescence emission can be used as fluorescent probes for understanding the role of ssDNA in various biological processes and to study various DNA-ligand interactions. Introduction The single strand DNA (ssDNA) forms an important inter- mediate in processes such as DNA replication, recombination, and repair. 1-5 ssDNA binding proteins play a major role in these processes and are known to interact specifically with ssDNA through π-stacking and electrostatic interactions. 6 Unlike the compact structure of the double strand DNA (dsDNA), ssDNA is flexible and is reported to exhibit sequence-dependent conformations and to exist as random coil or helical structures formed by the stacking interactions between the nucleobases. 7-10 Hence, it is of great interest to understand such conformational dynamics and to have an idea about the nature of interaction of small molecules with ssDNA. Besides this, the selective interactions of small molecules with ssDNA have great interest for biological and analytical purposes, for example, in DNA labeling, development of DNA based probes such as molecular beacons, detection of hybridization of single strands, and ssDNA detection and quantification. 11 The influence of steric and conformational factors on the extent of binding of the acridinium derivatives with dsDNA has been reported earlier. 12 It was observed that introduction of an ortho-substitution at the 9-phenyl group on the acridinium ring had profound influence on the intercalative interactions of the acridinium chromophore with dsDNA. This is because the nucleobases in dsDNA are situated well inside the double- stranded helix making it inaccessible to the ortho-methylphenyl substituted acridinium derivative leading to the negligible changes in its photophysical properties. However, the bases in ssDNA are more exposed to external medium 13 and the stacked bases should, in principle, favor the intercalation process. This led us to design and study the interactions of a few novel acridinium derivatives having bulky substituents with ssDNA with the objective of developing simple fluorescent probes which selectively interact with ssDNA when compared to dsDNA. Experimental Methods The equipment and procedure for spectral recordings are described elsewhere. 14-16 The fluorescence lifetimes were measured on an IBH picosecond single photon counting system using a 401 nm IBH NanoLED source and a Hamamatsu C4878- 02 MCP detector. The fluorescence decay profiles were decon- voluted using IBH Data Station software V2.1, fitted with a mono-, bi-, or triexponential decay and minimizing the  2 values of the fit to 1 ( 0.1. The fluorescence spectra were recorded on a SPEX-Fluorolog F112X spectrofluorimeter. The fluores- cence quantum yields were measured by the relative methods using optically dilute solutions, with 10-methylacridinium trifluoromethane sulfonate in water (Φ f ) 1) as the standard. 17 Laser flash photolysis experiments were carried out in Applied Photophysics model LKS-20 laser kinetic spectrometer using the third harmonic (355 nm) of a Quanta Ray GCR-12 series pulsed Nd:YAG laser. Circular dichroism (CD) spectra were recorded on Jasco Corporation, J-810 spectropolarimeter. Atomic force microscopy (AFM) images were recorded under ambient conditions using a Digital Instrument Multimode Nanoscope IV operating in the tapping mode regime. Microfabricated silicon cantilever tips (MPP-11100-10) with a resonance frequency of 299 kHz and a spring constant of 20-80 Nm -1 were used. The scan rate varied from 0.5 to 1 Hz. * To whom correspondence should be addressed. Tel: +91 471 2515362. Fax: +91 471 2490186 or +91 471 2491712. E-mail: d_ramaiah@rediffmail.com or rama@csrrltrd.ren.nic.in. 6549 J. Phys. Chem. B 2007, 111, 6549-6556 10.1021/jp071459j CCC: $37.00 © 2007 American Chemical Society Published on Web 05/22/2007