真核細胞的端粒，對於染色體尾端穩定性是相當重要的。在有單價陽離子如鈉或鉀離子存在之下，端粒尾端富含鳥嘌呤單股的DNA序列，可以藉由Hoogsteen氫鍵形成一個二級結構稱之為鳥嘌呤-四股結構。為了驗證人類端粒是否具有鳥嘌呤-四股結構的存在，我們利用了雙光子激發螢光生命期顯微技術，來尋找人體鼻咽癌細胞，中期染色體之中鳥嘌呤-四股結構的位置所在。然而，富含鳥嘌呤序列，可以具有多樣性鳥嘌呤-四股結構，而且改變環境條件可能使其結構互相轉換。舉例來說，鈉鉀離子交換後，會產生一個快速的光譜變化。我們在此利用數種方法，來瞭解鈉鉀離子交換之中所引起的快速光譜變化，其中所隱含的機制。螢光共振能量傳遞與單分子栓球實驗的研究，暗示著這個因鉀離子所產生的快速光譜變化，有可能不是F1UFF2，須經由一個完全展開的中間態。此外，變換溫度的圓二色光譜研究顯示，F1與F2之間的能障幾乎可以忽略。因此，我們認為這個鈉鉀離子交換所產生的快速光譜變化，是由於F1到F2之間，經過了快速的鹼基位移與環的重組所造成的。另一方面，我們在脫水的環境中觀察到，由鈉離子溶液之中的反平行鳥嘌呤-四股結構，轉換為鉀離子溶液中的平行鳥嘌呤-四股結構。利用van’t Hoff的方法，在熱解旋曲線之中，來估計各個摺疊狀態到完全展開狀態之間其自由能的差別，以及利用Eyring的方法，以即時變溫圓二色光譜，來估計鳥嘌呤-四股結構變化所需的活化能，嘗試建立一個HT22在鈉離子溶液中加入脫水環境(40% (w/v) PEG 200)的反平行鳥嘌呤-四股結構，轉換到鉀離子溶液中平行鳥嘌呤-四股結構的熱力學能量圖。此外，由於Cu2+可以誘導鳥嘌呤-四股結構的崩解，再者，EDTA2-可以抑制Cu2+離子的作用，使鳥嘌呤-四股結構可以由展開狀態變回摺疊狀態，根據此方法，我們發現動力學產物在人體生理條件下比較容易生成。更進一步，利用Cu2+離子在室溫下誘導鳥嘌呤-四股結構展開，來作為篩選鳥嘌呤-四股結構配位基的一個新方法。因此，我們篩選出3,6,9三端取代的BMVC4分子，可以作為之後研究的重心。

Telomeres, the ends of eukaryotic chromosomes, are essential for the stability of chromosomes. In the presence of monovalent cations such as Na+ or K+, the G-rich single stranded DNA of telomere can form a secondary structure through Hoogsteen hydrogen bonds, termed G-quadruplex (G4). We have applied two-photon excitation fluorescence lifetime microscope (2PE-FLIM) to successfully verify and map the localizations of G4 structures in human nasopharyngeal carcinoma metaphase chromosomes. In addition, the G-rich sequences can adopt various G4 structures and possibly interconvert among these structures upon changing solvent and temperature conditions. For example, a fast spectral conversion occurs under Na/K cation exchange. We have developed a number of methods to elucidate the mechanisms of this spectral conversion. Ensemble-based fluorescence resonance energy transfer (FRET) and single molecule tethered particle motion (TPM) studies suggested that the fast spectral conversion is unlikely due to F1UFF2 via a totally unfolded intermediate induced by potassium cations. In addition, temperature-dependent circular dichroism (CD) studies suggested that the energy barrier from F1 to F2 is almost negligible. Thus, we consider that the fast spectral conversion during Na/K cation exchange is due to F1F2 via rapid base shift and loop rearrangement. On the other hand, the structural conversion from the antiparallel G4 structure in Na+ solution to the parallel G4 structure in K+ solution was observed in the presence of dehydrated reagents.
Using thermodynamic and kinetic studies, a free energy diagram can be tentatively established for the structural conversion of HT22 from antiparallel form in Na+ solution to the parallel in K+ solution at 25℃ under 40 % (w/v) PEG 200 condition. It is known that the Cu2+ induces the unfolding of G4 structure while addition of the EDTA2- can chelate the Cu2+ to reverse the unfolded state to the folded state. Based on this and we found that the kinetic product is likely to play a major role in physiological condition.
Furthermore, G4 stabilizers are screened by a novel method based on Cu2+ -induced G4 unfolding at room temperature. Thus, 3,6,9 tri-substitution of BMVC4 core molecules are ready to prepare in further study.

(1) Greider, C. W.; Blackburn, E. H. Cell 1985, 43, 405.
(2) Moyzis, R. K.; Buckingham, J. M.; Cram, L. S.; Dani, M.; Deaven, L. L.; Jones, M. D.; Meyne, J.; Ratliff, R. L.; Wu, J. R. Proc Natl Acad Sci U S A 1988, 85, 6622.
(3) Greider, C. W.; Blackburn, E. H. Sci. Am. 1996, 274, 92.
(4) Williamson, J. R.; Raghuraman, M. K.; Cech, T. R. Cell 1989, 59, 871.
(5) Watson, J. D. Nat. New Biol. 1972, 239, 197.
(6) Harley, C. B.; Futcher, A. B.; Greider, C. W. Nature 1990, 345, 458.
(7) Goldstein, S. Science 1990, 249, 1129.
(8) Sandell, L. L.; Zakian, V. A. Cell 1993, 75, 729.
(9) Lundblad, V.; Szostak, J. W. Cell 1989, 57, 633.
(10) Morin, G. B. Cell 1989, 59, 521.
(11) Greider, C. W.; Blackburn, E. H. Cell 1987, 51, 887.
(12) Greider, C. W.; Blackburn, E. H. Nature 1989, 337, 331.
(13) Feng, J.; Funk, W. D.; Wang, S. S.; Weinrich, S. L.; Avilion, A. A.; Chiu, C. P.; Adams, R. R.; Chang, E.; Allsopp, R. C.; Yu, J.; et al. Science 1995, 269, 1236.
(14) Meyerson, M.; Counter, C. M.; Eaton, E. N.; Ellisen, L. W.; Steiner, P.; Caddle, S. D.; Ziaugra, L.; Beijersbergen, R. L.; Davidoff, M. J.; Liu, Q.; Bacchetti, S.; Haber, D. A.; Weinberg, R. A. Cell 1997, 90, 785.
(15) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 1994, 266, 2011.
(16) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. USA 1962, 48, 2013.
(17) Zahler, A. M.; Williamson, J. R.; Cech, T. R.; Prescott, D. M. Nature 1991, 350, 718.
(18) Mergny, J. L.; Helene, C. Nature Med. 1998, 4, 1366.
(19) Baumann, P.; Cech, T. R. Science 2001, 292, 1171.
(20) de Lange, T. Science 2009, 326, 948.
(21) Makarov, V. L.; Hirose, Y.; Langmore, J. P. Cell 1997, 88, 657.
(22) Wang, Y.; Patel, D. J. Structure 1993, 1, 263.
(23) Parkinson, G. N.; Lee, M. P. H.; Neidle, S. Nature 2002, 417, 876.
(24) Ambrus, A.; Chen, D.; Dai, J.; Bialis, T.; Jones, R. A.; Yang, D. Nucleic Acids Res. 2006, 34, 2723.
(25) Luu, K. N.; Phan, A. T.; Kuryavyi, V.; Lacroix, L.; Patel, D. J. J. Am. Chem. Soc. 2006, 128, 9963.
(26) Phan, A. T.; Luu, K. N.; Patel, D. J. Nucleic Acids Res. 2006, 34, 5715.
(27) Lim, K. W.; Amrane, S.; Bouaziz, S.; Xu, W. X.; Mu, Y. G.; Patel, D. J.; Luu, K. N.; Phan, A. T. J. Am. Chem. Soc. 2009, 131, 4301.
(28) Phan, A. T. FEBS J. 2010, 277, 1107.
(29) Lim, K. W.; Alberti, P.; Guedin, A.; Lacroix, L.; Riou, J. F.; Royle, N. J.; Mergny, J. L.; Phan, A. T. Nucleic Acids Res. 2009, 37, 6239.
(30) Davis, J. T. Angew. Chem. Int. Ed. 2004, 43, 668.
(31) Patel, D. J.; Phan, A. T.; Kuryavyi, V. Nucleic Acids Res. 2007, 35, 7429.
(32) Neidle, S. Curr. Opin. Struct. Biol. 2009, 19, 239.
(33) Hud, N. V.; Smith, F. W.; Anet, F. A.; Feigon, J. Biochemistry 1996, 35, 15383.
(34) Ying, L.; Green, J. J.; Li, H.; Klenerman, D.; Balasubramanian, S. Proc. Natl. Acad. Sci. USA 2003, 100, 14629.
(35) Lee, J. Y.; Okumus, B.; Kim, D. S.; Ha, T. Proc. Natl. Acad. Sci. USA 2005, 102, 18938.
(36) N.V. Hud, and J. Plavec. The role of cations in determining quadruplex structure and stability. In Quadruplex Nucleic Acids.; S.Neidle, S. Balasubramanian, Editors; Royal Society of Chemistry, Cambridge, UK, 2006; pp 100.
(37) Chang, C. C.; Chien, C. W.; Lin, Y. H.; Kang, C. C.; Chang, T. C. Nucleic Acids Res. 2007, 35, 2846.
(38) Cantor, C. R.; Warshaw, M. M.; Shapiro, H. Biopolymers 1970, 9, 1059.
(39) Chang, C. C.; Wu, J. Y.; Chang, T. C. J. Chin. Chem. Soc. 2003, 50, 185.
(40) 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.
(41) Oster, G.; Nishijima, Y. J. Am. Chem. Soc. 1956, 78, 1581.
(42) 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.
(43) Rotkiewi.K; Grellman.Kh; Grabowsk.Zr. Chem. Phys. Lett. 1973, 19, 315.
(44) Nag, A.; Bhattacharyya, K. Chem. Phys. Lett. 1990, 169, 12.
(45) Rettig, W. Angew. Chem. Int. Ed. 1986, 25, 971.
(46) Chang, C. C.; Chu, J. F.; Kuo, H. H.; Kang, C. C.; Lin, S. H.; Chang, T. C. J. Lumin. 2006, 119, 84.
(47) Castex, M. C.; Olivero, C.; Pichler, G.; Ades, D.; Cloutet, E.; Siove, A. Synthetic Metals 2001, 122, 59.
(48) Ades, D.; Boucard, V.; Cloutet, E.; Siove, A.; Olivero, C.; Castex, M. C.; Pichler, G. J. Appl. Phys. 2000, 87, 7290.
(49) Changenet, P.; Zhang, H.; van der Meer, M. J.; Glasbeek, M.; Plaza, P.; Martin, M. M. J. Phys. Chem. A 1998, 102, 6716.
(50) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73.
(51) Thompson, R. E.; Larson, D. R.; Webb, W. W. Biophys. J. 2002, 82, 2775.
(52) Albota, M.; Beljonne, D.; Bredas, J. L.; Ehrlich, J. E.; Fu, J. Y.; Heikal, A. A.; Hess, S. E.; Kogej, T.; Levin, M. D.; Marder, S. R.; McCord-Maughon, D.; Perry, J. W.; Rockel, H.; Rumi, M.; Subramaniam, G.; Webb, W. W.; Wu, X. L.; Xu, C. Science 1998, 281, 1653.
(53) Chang, C. C.; Chu, J. F.; Kao, F. J.; Chiu, Y. C.; Lou, P. J.; Chen, H. C.; Chang, T. C. Anal. Chem. 2006, 78, 2810.
(54) Knemeyer, J. P.; Herten, D. P.; Sauer, M. Anal. Chem. 2003, 75, 2147.
(55) Xu, Y.; Noguchi, Y.; Sugiyama, H. Bioorg. Med. Chem. 2006, 14, 5584.
(56) Vorlickova, M.; Kejnovska, I.; Tumova, M.; Kypr, J. Eur. Biophys. J. 2001, 30, 179.
(57) Phan, A. T.; Kuryavyi, V.; Luu, K. N.; Patel, D. J. Nucleic Acids Res. 2007, 35, 6517.
(58) Gray, R. D.; Li, J.; Chaires, J. B. J. Phys. Chem. B 2009, 113, 2676.
(59) Weiss, S. Science 1999, 283, 1676.
(60) Smith, D. E.; Babcock, H. P.; Chu, S. Science 1999, 283, 1724.
(61) Moerner, W. E.; Orrit, M. Science 1999, 283, 1670.
(62) Gelles, J.; Schnapp, B. J.; Sheetz, M. P. Nature 1988, 331, 450.
(63) Yin, H.; Landick, R.; Gelles, J. Biophys. J. 1994, 67, 2468.
(64) Yin, H.; Artsimovitch, I.; Landick, R.; Gelles, J. Proc. Natl. Acad. Sci. USA 1999, 96, 13124.
(65) Dohoney, K. M.; Gelles, J. Nature 2001, 409, 370.
(66) Schafer, D. A.; Gelles, J.; Sheetz, M. P.; Landick, R. Nature 1991, 352, 444.
(67) Mumm, J. P.; Landy, A.; Gelles, J. EMBO J. 2006, 25, 4586.
(68) Guerra, R. F.; Imperadori, L.; Mantovani, R.; Dunlap, D. D.; Finzi, L. Biophys. J. 2007, 93, 176.
(69) Pouget, N.; Turlan, C.; Destainville, N.; Salome, L.; Chandler, M. Nucleic Acids Res. 2006, 34, 4313.
(70) Fan, H. F.; Li, H. W. Biophys. J. 2009, 96, 1875.
(71) Finzi, L.; Gelles, J. Science 1995, 267, 378.
(72) Lin, C. T.; Tseng, T. Y.; Wang, Z. F.; Chang, T. C. J. Phys. Chem. B 2011, 115, 2360.
(73) Miura, T.; Benevides, J. M.; Thomas, G. J., Jr. J. Mol. Biol. 1995, 248, 233.
(74) Gray, D. M.; Wen, J. D.; Gray, C. W.; Repges, R.; Repges, C.; Raabe, G.; Fleischhauer, J. Chirality 2008, 20, 431.
(75) Masiero, S.; Trotta, R.; Pieraccini, S.; De Tito, S.; Perone, R.; Randazzo, A.; Spada, G. P. Org. Biomol. Chem. 2010, 8, 2683.
(76) Li, W.; Miyoshi, D.; Nakano, S.; Sugimoto, N. Biochemistry 2003, 42, 11736.
(77) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. USA 1967, 58, 719.
(78) Green, J. J.; Ying, L.; Klenerman, D.; Balasubramanian, S. J. Am. Chem. Soc. 2003, 125, 3763.
(79) Fegan, A.; Shirude, P. S.; Ying, L.; Balasubramanian, S. Chem. Commun. 2010, 46, 946.
(80) Zhang, Z.; Dai, J.; Veliath, E.; Jones, R. A.; Yang, D. Nucleic Acids Res. 2010, 38, 1009.
(81) Chu, J. F.; Chang, T. C.; Li, H. W. Biophys. J. 2010, 98, 1608.
(82) Sen, D.; Gilbert, W. Nature 1988, 334, 364.
(83) Raghuraman, M. K.; Cech, T. R. Nucleic Acids Res. 1990, 18, 4543.
(84) Miller, M. C.; Buscaglia, R.; Chaires, J. B.; Lane, A. N.; Trent, J. O. J. Am. Chem. Soc. 2010, 132, 17105.
(85) Heddi, B.; Phan, A. T. J. Am. Chem. Soc. 2011, dx.doi.org/10.1021/ja200786q.
(86) Miyoshi, D.; Karimata, H.; Sugimoto, N. J. Am. Chem. Soc. 2006, 128, 7957.
(87) Xue, Y.; Kan, Z. Y.; Wang, Q.; Yao, Y.; Liu, J.; Hao, Y. H.; Tan, Z. J. Am. Chem. Soc. 2007, 129, 11185.
(88) Xu, L.; Feng, S.; Zhou, X. Chem. Commun. 2011, 47, 3517.
(89) Renciuk, D.; Kejnovska, I.; Skolakova, P.; Bednarova, K.; Motlova, J.; Vorlickova, M. Nucleic Acids Res. 2009, 37, 6625.
(90) Lim, K. W.; Lacroix, L.; Yue, D. J.; Lim, J. K.; Lim, J. M.; Phan, A. T. J. Am. Chem. Soc. 2010, 132, 12331.
(91) Olsen, C. M.; Lee, H. T.; Marky, L. A. J. Phys. Chem. B 2009, 113, 2587.
(92) Antonacci, C.; Chaires, J. B.; Sheardy, R. D. Biochemistry 2007, 46, 4654.
(93) Eyring, H. Chemical Reviews 1935, 17, 65.
(94) Miyoshi, D.; Nakao, A.; Sugimoto, N. Nucleic Acids Res 2003, 31, 1156.
(95) Monchaud, D.; Yang, P.; Lacroix, L.; Teulade-Fichou, M. P.; Mergny, J. L. Angew. Chem. Int. Ed. 2008, 47, 4858.
(96) Zaug, A. J.; Podell, E. R.; Cech, T. R. Proc. Natl. Acad. Sci. USA 2005, 102, 10864.
(97) Gomez, D.; Guedin, A.; Mergny, J. L.; Salles, B.; Riou, J. F.; Teulade-Fichou, M. P.; Calsou, P. Nucleic Acids Res. 2010, 38, 7187.
(98) Sanders, C. M. Biochem. J. 2010, 430, 119.
(99) Miyoshi, D.; Pramanik, S.; Nakamura, K.; Usui, K.; Nakano, S.; Saxena, S.; Matsui, J.; Sugimoto, N. Chem. Commun. 2011, 47, 2790.
(100) McLuckie, K. I.; Waller, Z. A.; Sanders, D. A.; Alves, D.; Rodriguez, R.; Dash, J.; McKenzie, G. J.; Venkitaraman, A. R.; Balasubramanian, S. J. Am. Chem. Soc. 2011, 133, 2658.
(101) Hansel, R.; Lohr, F.; Foldynova-Trantirkova, S.; Bamberg, E.; Trantirek, L.; Dotsch, V. Nucleic Acids Res. 2011, doi:10.1093/nar/gkr174.