G-Quadruplex DNA Binding by a Series of Carbocyanine Dyes Sean M. Kerwin,* Daekyu Sun, Jonathan T. Kern, Anupama Rangan and Pei Wang Thomas Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, USA Received 9 May 2001; accepted 6 June 2001 Abstract—We have examined a number of carbocyanine dyes for their ability to bind intramolecular G-quadruplex DNA structures (G4 0 -DNA) using a Taq polymerase stop assay. Of the five dyes examined, only one, N,N’-diethylthiacarbocyanine iodide (DTC), was found to bind to G4 0 -DNA. DTC was also the only dye found to inhibit human telomerase at 50 mM concentration. # 2001 Elsevier Science Ltd. All rights reserved. Telomerase is present in the vast majority of cancer cells and largely absent in normal somatic tissue, and is thought to play an important role in the maintenance of telomeres in cancer cells. Inhibitors of telomerase are being examined as potential anticancer agents. 1,2 One such class of telomerase inhibitors are compounds that bind to G-quadruplex DNA structures. These four- stranded DNA structures are formed from a variety of G-rich DNA sequences through the association of gua- nosine residues into G-tetrads (Fig. 1A). 3 In particular, telomeric DNA sequences [e.g., 5 0 -d(TTAGGG) n in humans] have been shown to form four-stranded G-quad- ruplex structures in vitro. 4,5 There are three major classes of G-quadruplex structures (Fig. 1B): those formed from four separate G-rich DNA strands (G4-DNA), those formed from two DNA strands (G 0 2-DNA), and those formed from a single strand of DNA (G4 0 -DNA). Although ligands that bind to G-quadruplex DNA have been shown to inhibit telomerase, 3,6 8 there has not been any attempt to relate the selectivity of these ligands for the different forms of G-quadruplex DNA such as G4-, G 0 2- and G4 0 -DNA with their telomerase inhibitory properties. Here, we report our studies of the G-quadruplex DNA binding and human telomerase inhibition properties of a series of carbocyanine dyes. These data demonstrate the importance of G4 0 -DNA binding in the ability of G-quadruplex DNA ligands to inhibit telomerase. Shafer and co-workers have previously reported the G- quadruplex DNA binding by the carbocyanine DODC. 9 They characterized the interaction of DODC with a variety of G-quadruplex and duplex DNA structures by fluorescence, absorption, and CD spectroscopy, as well as satellite hole spectroscopy. 10 DODC interacts speci- fically with G 0 2-DNA structures. Binding constants determined from absorption spectroscopic titrations reveal that the binding affinity of DODC for the G 0 2- DNA form of [d(G 4 T 4 G 4 )] 2 is 5 times higher than for double-stranded DNA. Equilibrium dialysis experi- ments, however, demonstrate a preference for DODC binding to triplex DNA over G-quadruplex DNA. 11 We examined a series of carbocyanine dyes for their ability to interact with G4 0 -DNA structures using the 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00490-5 Bioorganic & Medicinal Chemistry Letters 11 (2001) 2411–2414 Figure 1. (A) Structure of a G-tetrad; (B) different forms of G-quad- ruplex DNA. *Corresponding author. Tel.: +1-512-471-5074; fax: +1-512-232- 2606; e-mail: skerwin@mail.utexas.edu