Possibility of an Antiparallel (Tetramer) Quadruplex Exhibited by the Double Repeat of the Human Telomere ² Mahima Kaushik, Aparna Bansal, Sarika Saxena, and Shrikant Kukreti* Nucleic Acids Research Lab, Department of Chemistry, UniVersity of Delhi, (North Campus), Delhi 110007, India ReceiVed October 9, 2006; ReVised Manuscript ReceiVed April 6, 2007 ABSTRACT: Under physiological concentrations of Na + and K + , human telomeric DNA can self-associate into G-quadruplexes. On the basis of circular dichroism, gel electrophoresis, gel filtration, and UV-melting experiments, we report here that the double repeat of human telomere (d-TTAGGGTTAGGG; HUM2) forms parallel as well as antiparallel quadruplexes in the presence of K + , whereas Na + facilitates only the antiparallel form. Here, the gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. By correlating the gel and CD experiments, the antiparallel G-quadruplex was identified as a tetrameric species, whereas the parallel G-quadruplex was found to be dimeric. Both structural species were separated through gel filtration, which when run on native polyacrylamide gel electrphoresis (PAGE), confirmed their molecularity. UV-melting profiles also confirm the presence of two biphasic and one monophasic structural species in the presence of K + and Na + , respectively. Though our observation is consistent with the recent NMR report (Phan, A. T., and Patel, D. J. (2003) J. Am. Chem. Soc. 125, 15021-15027), it seems to differ in terms of the molecularity of the antiparallel quadruplex. A model is proposed for an antiparallel tetrameric quadruplex, showing the possibility of Watson-Crick hydrogen bonds between intervening bases on antiparallel strands. This article expands the known structural motifs of DNA quadruplexes. To the best of our knowledge, four-stranded antiparallel quadruplexes have not been characterized to date. On the basis of the model, we hypothesize a possible mechanism for telomere-telomere association involving their G-overhangs, during certain stages of the cell cycle. The knowledge of peculiar geometries of the G-quadruplexes may also have implications for its specific recognition by ligands. Telomeres are specialized structures comprising DNA and protein, capping the ends of eukaryotic chromosomes. The possible functions of telomeres include maintaining the structural integrity of chromosomes, ensuring complete replication of their extreme ends, and helping to establish the three-dimensional architecture of the nucleus and/or chromosome pairing (1-3). The G-rich strand of the telomere has a single-stranded extension toward the 3′-end and can be written in a general form as G n (A/T) m when n > 1 and m ) 1-4. The resulting single-stranded overhang, or telomere tail, is capable of forming complex intrastranded associations (4). In addition to telomere ends of eukaryotic chromosomes, runs of G’s may also be found in other locations including the c-myc promoter (5), the triplet repeat region that can cause a variety of neurological disorders (6), the recombination and mutation hot spots (7), and the switch region of immunoglobins (8). Telomerase, a ribonucleoprotein complex which ensures replication of the telomeres, may be proposed as attractive targets for the discovery of new anticancer agents (9). A number of small molecules have been discovered to inhibit the function of telomerase by stabilizing G-quadruplex structures (10-11). Davis (12) has elegantly reviewed the significance of G-quartet assemblies in areas ranging from structural biology and medicinal chemistry to supramolecular chemistry and nanotechnology. It is now well established that the G-rich sequences can self-associate in Vitro to form different types of DNA quadruplexes, all containing guanine base tetrads or quartets (G-tetrad); planar structures composed of four Hoogsteen base paired guanines in a cyclic array, thanks to the multiple hydrogen bonding donor and acceptor sites of the nucleobase guanine, which makes it “sticky” (12). Many laboratories have reported that DNA oligonucleotides having repetitive tracts of guanine bases can form G-quadruplex structures that display an amazing polymorphism under cellular envi- ronmental conditions, such as pH, cations, and temperature. Monovalent cations, notably K + and Na + , greatly stabilize G-quadruplex structures presumably by coordinating with eight carbonyl oxygens sandwiched between two coplanar quartets. The structure of synthetic oligonucleotides corre- sponding to telomeric G-rich strands has been discussed extensively in recent, excellent reviews (13-16). Several structures have been identified, and these include four- stranded intermolecular quadruplexes, hairpin dimers, and monomolecular intramolecularly folded quadruplexes (14, 17, 18). There are a number of ways in which four strands of DNA may interact and pair into a stable structure. They ² This work was supported by grants-in aid (no.F.12-28/2002 (SR- 1) from the University Grant Commission, New Delhi. A.B. is a recipient of a Junior Research Fellowship from UGC. * To whom correspondence should be addressed. Tel: (011) 27666726. Fax: +91 11 27666605. E-mail: skukreti@chemistry.du.ac.in or kukretishrikant@yahoo.com. 7119 Biochemistry 2007, 46, 7119-7131 10.1021/bi0621009 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/25/2007