2006 Chem. Commun., 2012, 48, 2006–2008 This journal is c The Royal Society of Chemistry 2012 Cite this: Chem. Commun., 2012, 48, 2006–2008 Intramolecular folding in three tandem guanine repeats of human telomeric DNAw Deepak Koirala, a Tomoko Mashimo, b Yuta Sannohe, b Zhongbo Yu, a Hanbin Mao* a and Hiroshi Sugiyama* bcd Received 1st November 2011, Accepted 13th December 2011 DOI: 10.1039/c2cc16752b Intramolecular folding in three tandem guanine repeats of human telomeric DNA has been investigated using optical- tweezers, MD simulation and circular dichroism. A mechanically and thermodynamically stable species in this sequence shows a structure consistent with a triplex conformation. A similar species has also been observed to coexist with a G-quadruplex in a DNA sequence with four tandem guanine repeats. Telomeres of eukaryotic chromosomes play a vital role in maintaining gene integrity and cell growth. 1 In normal cells, the telomere is shortened each time a chromosome is replicated during cell division, eventually leading to apoptosis. 2 However, majority of cancer cells maintain the length of the telomere due to enhanced activity of telomerase. 3 Human telomeres have the propensity to form intramolecular G-quadruplex (GQ) structures in the 3 0 overhang of the region. 4 A GQ is a tetraplex structure which is composed of a stack of G-quartets, 5 each of which contains a quadrilateral of four guanines (G) held together by Hoogsteen base pairs and stabilized by monovalent cations such as K + or Na + . 6 It has been observed that small-molecules such as telomestatin, 7 quarfloxin, 8 and pyridostatin 9,10 effectively stabilize the GQ structures and reduce the telomerase activity. This implied a potential regulatory function for GQ to maintain the length of telomeres. The structural diversity has been well recorded for GQs in four tandem guanine repeats of the human telomeric DNA. 11 However, the possibility of forming alternative structures in such sequences has not been explored well. Single-molecule investigations by Balasubramanian et al. 12 and Ha et al. 13 have revealed unknown species other than GQs in the human telomeric DNA. Independent studies by Yang et al., 14 Sugiyama et al., 15 and Chaires et al. 16 have proposed that these structures could be GQ intermediates that employ only three tandem guanine repeats to assume a triplex structure. NMR structure of a 3 + 1 bimolecular GQ has supported these proposals. 17 Nevertheless, stand-alone G-triplex structures have not been observed directly in three tandem guanine repeats. Using an optical-tweezer (OT) based single-molecule assay in assistance with molecular dynamics (MD) simulation and circular dichroism (CD), here, we have investigated the folded structures formed in the human telomeric sequences with three G-rich repeats, TTA(GGGTTA) 3 (Tel-3G), and four G-rich repeats, TTA(GGGTTA) 4 (Tel-4G). We have observed mecha- nically and thermodynamically stable species different from the GQ in both sequences. In addition, we have demonstrated that such intramolecularly folded species in the Tel-3G or Tel-4G are consistent with the triplex conformation. These findings depict a complex folding pattern in human telomeres. The mechanical stabilities of these structures revealed by OTs are of unique importance to elaborate their potential interactions with telomerase, a motor protein that generates a load force. First, we used CD to investigate the folded structures formed in the Tel-3G and Tel-4G in a 10 mM Tris buffer (pH 7.4) containing 100 mM Na + . As shown in Fig. 1a, the CD spectrum of the Tel-4G has two peaks at 293 and 250 nm with a valley at 271 nm, which is consistent with previous findings that human telomeric sequence prefers a basket type GQ in this buffer. 14 The CD spectrum of the Tel-3G, however, shows dramatically different features with a pronounced peak at 257 nm and a valley at 238 nm. In addition, a small valley and a peak were observed at 280 nm and 295 nm respectively. These Tel-3G features are consistent with previous observa- tions on similar sequences that suggested the presence of intramolecularly folded structures other than GQ. 18 When temperature was raised, we observed the dissolution of all these features except the 238 nm valley. At the same time, a broad peak of 275 nm appeared (Fig. 1b), which is consistent with the spectrum of unstructured ssDNA. 19 A plot of the 257 nm peak intensity vs. temperature reveals a melting transition with T m of 55 Æ 1 1C at 10 mM sample concentration (Fig. 1c). This result indicated the formation of a stable structure in the Tel-3G. As demonstrated in Fig. 1d, T m of 3–30 mM of Tel-3G is constant, indicating that the folded structure in the Tel-3G is indeed of intramolecular nature in this concentration range. a Department of Chemistry & Biochemistry, Kent State University, Kent, OH 44242, USA. E-mail: hmao@kent.edu b Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan c Institute for Integrated Cell Material Sciences (iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan d CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. E-mail: hs@kuchem.kyoto-u.ac.jp w Electronic supplementary information (ESI) available: Experimental details, calculations and supplementary figures. See DOI: 10.1039/ c2cc16752b ChemComm Dynamic Article Links www.rsc.org/chemcomm COMMUNICATION Downloaded by KENT STATE UNIVERSITY on 22 August 2012 Published on 14 December 2011 on http://pubs.rsc.org | doi:10.1039/C2CC16752B View Online / Journal Homepage / Table of Contents for this issue