Verification of Antiparallel G-Quadruplex Structure in Human Telomeres by Using Two-Photon Excitation Fluorescence Lifetime Imaging Microscopy of the 3,6-Bis(1-methyl-4-vinylpyridinium)carbazole Diiodide Molecule Cheng-Chung Chang, † Jen-Fei Chu, †,‡ Fu-Jen Kao, §,| Yi-Chun Chiu, | Pei-Jen Lou, ⊥ Huei-Chin Chen, ⊥ and Ta-Chau Chang* ,†,§ Institute of Atomic and Molecular Sciences, Academia Sinica, P. O. Box 23-166, Taipei, 106, Taiwan, Republic of China, Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan, Republic of China, Institute of Biophotonics Engineering, National Yang-Ming University, Taipei, 11221, Taiwan, Republic of China, Institute of Electro-Optical Engineering, National Yat-sen University, Kaohsiung, 80424, Taiwan, Republic of China, and Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China Different G-quadruplex structures for the human telom- eric sequence d(T 2 AG 3 ) 4 in vitro have been documented in the presence of sodium and potassium. Verification of the G-quadruplex structures in human telomeres in vivo is the main issue in establishing the biological function of the G-quadruplex structures in telomeres as well as the development of anticancer agents. Here we have applied two-photon excitation fluorescence lifetime imaging mi- croscopy to measure the fluorescence lifetime of the BMVC molecule upon interaction with various DNA structures. The distinction in lifetime measured with submicrometer spatial resolution in two-photon excitation fluorescence lifetime imaging microscopy provides a powerful approach not only to verify the existence of the antiparallel G-quadruplex structure in human telomeres but also to map its localizations in metaphase chromo- somes. Telomeres, the ends of chromosomes, are essential for genome integrity and chromosome replication. 1,2 Telomeres normally contain tandem repeats of guanine-rich (G-rich) motifs, for example, the hexameric repeats of TTAGGG/CCCTAA in verte- brate telomeres. Of special interest is that the 3′-overhang G-rich single strand with 50-200 bases could adopt G-quadruplex structures under physiological conditions. Since the folding of telomeric DNA into a G-quadruplex structure has been shown to inhibit telomerase activity in vitro, molecules that stabilize G- quadruplex structures have the potential to interfere with telomere replication and possibly to serve as anticancer agents. 3-5 Although direct evidence for the presence of G-quadruplex structures in vivo has been reported in the cilitate Stylonychia, 6 the promoter of c-myc, 7 and the human telomeres, 8 the existence of G- quadruplex structures in human cells is still in debate. 9 To distinguish the very small amounts of G-quadruplex structures from the overwhelming amounts of duplex structures in chromosomes, a 3,6-bis(1-methyl-4- vinylpyridinium)carbazole diiodide (BMVC, U.S. patent 2005-0090671) molecule was syn- thesized to recognize the unimolecular G-quadruplex structure of human telomeric sequence of d(T 2 AG 3 ) 4 (H24). 10 Chart 1A shows the chemical structure of BMVC. Significant increase of fluorescence yield and distinctive fluorescence properties of the BMVC upon binding to various DNA structures could allow us to verify the presence of G-quadruplex structures in human telom- eres. 8 However, the localization of the G-quadruplex structures in metaphase chromosomes was not directly visualized. Moreover, two types of G-quadruplex structures of the human telomeres have been documented in vitro. 11-14 Structures B and C in Chart I show two different G-quadruplex structures of d[AG 3 (T 2 AG 3 ) 3 ] (H22) * To whom correspondence should be addressed. E-mail: tcchang@ po.iams.sinica.edu.tw. † Academia Sinica. ‡ National Taiwan Normal University. § National Yang-Ming University. | National Yat-sen University. ⊥ National Taiwan University Hospital. (1) Blackburn, E. H.; Greider, C. W. Telomeres; Cold Spring Harbor Laboratory Press: New York, 1996. (2) Williamson, J. R. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 703-730. (3) Mergny, J. L.; He ´le `ne, C. Nat. Med. 1998, 4, 1366-1367. (4) Kerwin, S. M. Curr. Pharm. Des. 2000, 6, 441-471. (5) Han, H.; Hurley, L. H. Trends Pharmacol. Sci. 2001, 21, 136-142. (6) Schaffitzel, C.; Berger, I.; Postberg, J.; Hanes, J.; Lipps, H. J.; Plu ¨ckthun, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8572-8577. (7) Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11593-11598. (8) Chang, C. C.; Kuo, I.-C.; Ling, I.-F.; Chen, C. T.; Chen, H. C.; Lou, P. J.; Lin, J. J.; Chang, T.-C. Anal. Chem. 2004, 76, 4490-4494. (9) Granotier, C.; Pennarun, G.; Riou, L.; Hoffschir, F.; Gauthier, L. R.; Cian, A. D.; Gomez, D.; Mandine, E.; Riou, J.-F.; Mergny, J.-L.; Mailliet, P.; Dutrillaux, B.; Boussin, F. D. Nucleic Acids Res. 2005, 33, 4182-4190. (10) Chang, C. C.; Wu, J. Y.; Chien, C. W.; Wu, W. S.; Liu, H.; Kang, C. C.; Yu, L. J.; Chang, T.-C. Anal. Chem. 2003, 75, 6177-6183. (11) Wang, Y.; Patel, D. J. Structure 1993, 1, 262-283. (12) Neidle, S.; Parkinson, G. N. Curr. Opin. Struct. Biol. 2003, 13, 275-283. Anal. Chem. 2006, 78, 2810-2815 2810 Analytical Chemistry, Vol. 78, No. 8, April 15, 2006 10.1021/ac052218f CCC: $33.50 © 2006 American Chemical Society Published on Web 03/15/2006