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Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin
Spectrochimica Acta Part A 74 (2009) 243–247
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin Huijuan Zhang a,∗ , Xiao Xiao a , Peng Wang b , Siping Pang a , Feng Qu a , Xicheng Ai b,∗∗ , Jianping Zhang b a b
School of Life Science and Technology, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 100081, China Department of Chemistry, Renmin University of China, Beijing 100872, China
a r t i c l e
i n f o
Article history: Received 29 November 2008 Received in revised form 28 May 2009 Accepted 7 June 2009 This paper is dedicated to professor Yongzhong Yu on the occasion of his 85th birthday. Keywords: G-quadruplex (G4) TMPyP4 Conformational conversion Spectroscopy
a b s t r a c t The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)2 (S12) have been investigated by means of circular dichroism (CD), UV–visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K+ , and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K+ . Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)3 T) in the absence of K+ show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Guanine-rich sequences exist in many important DNA regions such as telomeres, some oncogenes, immunoglobulin switch regions and recombination hot spots [1–4]. Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG), which can form four-stranded structures called G-quadruplexes (G4) in the presence of K+ or Na+ [5]. The formation of the G4 structures has been shown to decrease the activity of telomerase, which is responsible for elongating telomeres [6]. Therefore, seeking for efficient telomerase inhibitors that are capable of inducing and stabilizing the G4 structure currently attracts great attention in the field of chemotherapy. For the well-matched molecular dimension as well as the strong – stacking and electrostatic interactions, the binding of the cationic porphyrin, meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4), with DNA G4 has been subjected to extensive investigations [7–17]. Many investigations have been focused on the structure of TMPyP4–G4 complex, the binding modes and the binding stoichiometries [14–16], however, the conformational conversion of G4 structure induced by TMPyP4 has not been concerned much in the last decade.
∗ Corresponding author. Tel.: +86 10 6891 2140; fax: +86 10 6891 4607. ∗∗ Corresponding author. Tel.: +86 10 6251 6604; fax: +86 10 6251 6444. E-mail addresses: [email protected] (H. Zhang), [email protected] (X. Ai). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.06.018
In our previous study, we have investigated the interaction between TMPyP4 and G4 formed from the single-strand d(TAGGG(TTAGGG)3 T) (S24) DNA, and firstly found that the conformation of G4 could be transformed by the induction of TMPyP4 [18]. Here we studied the conformational conversion of G4 from d(TTAGGG)2 (S12) DNA induced by TMPyP4 in the presence and absence of K+ . Moreover, comparable experiments of TMPyP4 with S24 and d(TTAGGG) (S6) were made in the absence of K+ , which indicated the important role of the loops for the formation of G4. As there is still no direct evidence for the accurate structure of G4 in vivo, it is of great biological significance to investigate the singlestrand telomeric DNA of S12, S24 and S6 with different length. For the case of S12 DNA, it represents the condition that G4 is formed from two single-strand DNA of two sister chromosomes. For the case of S24 DNA, it represents that G4 is formed from a singlestrand DNA of one chromosome. And for S6, it represents that G4 is formed from four single-strand DNA, which is a simplified model for investigation. 2. Materials and methods 2.1. Sample preparation Oligonucleotides S12, S6, and S24 DNA (PAGE-purified, >99% homogeneous) were purchased from Bioasia Biologic Technology Co. Ltd., and were used without further purification. The solution of DNA was prepared in Tris–HCl buffer (10 mM, pH = 7.2) in the presence of 0 and 10 mM potassium chloride, respectively, and was
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quantitated by the use of UV–visible spectrophotometry at 260 nm with an extinction coefficient of ε260 = 2.29 × 105 M cm−1 per G4 as estimated by using the nearest-neighbor method [19]. TMPyP4 was synthesized following the procedures described in Refs. [20,21]. Fresh solution used in the spectroscopic titration was prepared in the same buffer as those used for DNA, and were quantitated by using ε422 = 2.26 × 105 M−1 cm−1 . To avoid photodegradation, the preparations were kept in the dark before the spectroscopic titration experiments. For the convenience of data analysis, the molar ratio of TMPyP4to-DNA was defined as R in this study. 2.2. Circular dichroism titration The formation of G4 and the induced optical activity of cationic TMPyP4 were monitored by the use of circular dichroism (CD) spectroscopy. The CD spectra were recorded on a CD spectrophotometer (CD6, Jobin-Yvon, Longjumeau, France) and were corrected against the background of buffer. The CD spectra of DNA were examined over 220–325 nm, and those of TMPyP4–DNA complex over 220–500 nm. 2.3. UV–visible absorption and fluorescence titration The binding of TMPyP4 to S12 DNA was monitored by the use of steady-state UV–visible absorption and fluorescence spectroscopies. The final concentration of TMPyP4 was fixed at 4.6 × 10−6 M, to which the annealed DNA solution was added gradually to make a final concentration of S12 in the range of 0–4.7 × 10−6 M (per G4). After incubation at 25 ◦ C for 5 min, the absorption spectrum of each mixture was recorded from 350 nm to 700 nm on a spectrometer (U-3310, Hitachi, Hitachinaka, Japan). Fluorescence spectra of TMPyP4 in the same series of preparation as used in absorption titration were recorded under the photoexcitation at 400 nm on a spectrophotometer (F-2500, Hitachi, Hitachinaka, Japan). 3. Results and discussion 3.1. Conformational conversion of the parallel G4 to the mixed parallel/antiparallel structure from S12 DNA induced by TMPyP4 in the presence of K+ The conformational change of the G4 structure induced by TMPyP4 was examined by means of CD spectroscopy in the presence of K+ (Fig. 1(a)). The solid line in Fig. 1(a) shows the CD spectrum of S12 in Tris–HCl buffer (free from TMPyP4) after annealing treatment, which is characterized by a strong positive peak at 260 nm and a negative one at 239 nm. These features are ascribable to a parallel G4 structure formed from the single-strand S12 DNA in the presence of K+ , which is in accordance with the crystal structure reported by Parkinson et al. recently [14]. With the addition of TMPyP4, the change of the CD spectra of the parallel G4 from S12 are presented in Fig. 1(a) as the dotted line (R = 2.3) and the dashed line (R = 9.2). The inset shows the CD intensity plotted against R at characteristic wavelengths of 260 nm, 290 nm and 440 nm. The addition of TMPyP4 causes apparent decrease of the 260-nm peak and the occurrence of the positive signal at 290 nm until almost keeps constant when R ≈ 5. Meanwhile, a strong negative induced CD peak of porphyrin at 440 nm appears. These changes indicate the transformation of the G4 structure from the parallel type to the mixed parallel/antiparallel one with the binding of TMPyP4 molecules. After R exceeds 5, the CD signal of DNA and TMPyP4 almost keeps constant. The turning point of the titration process that occurs at R ≈ 5, can be regarded as the
saturated binding number of TMPyP4 to induce the G4 structural conversion. The interaction of TMPyP4 and parallel G4 was further investigated by absorption and fluorescence spectroscopy. Fig. 2(a) shows the UV–visible absorption spectra of TMPyP4 (4.6 × 10−6 M) titrated with G4 DNA (0–4.7 × 10−6 M). Significant bathochromic shift (14 nm) and hypochromicity (41%) of the Soret band were observed owing to the strong TMPyP4–G4 interaction. Fig. 3(a) shows the corresponding fluorescence spectra of TMPyP4 titrated with G4 DNA under the excitation wavelength of 400 nm. When free from DNA, TMPyP4 is moderately fluorescent with a fluorescence quantum yield of 0.05. The fluorescence spectrum of whom is characterized by a broad dual-band pattern in the range of 600–800 nm. Upon adding S12, the fluorescence intensity decreases and two peaks at 663 nm and 724 nm become better resolved. The decrease in intensity can be attributed to the quench of singlet excited-state TMPyP4 owing to the electron transfer reaction involving groundstate guanine as the electron donor [22]. Taking together above results, it is shown that TMPyP4 interacts strongly with G4 formed from S12 in the presence of K+ , corresponding to a conformational transition of G4 from the parallel type to the mixed parallel/antiparallel one within a TMPyP4to-DNA ratio of 5. Additionally, to get more information about the G4 conformation, NMR experiment is under study in our laboratory. 3.2. Formation of antiparallel G4 from the single-strand S12 DNA induced by TMPyP4 in the absence of K+ The solid line in Fig. 1(b) shows the CD spectrum of S12 in Tris–HCl buffer (free from both K+ and TMPyP4), which is characterized by a strong positive peak at 256 nm and a weak negative one at 238 nm. These features are ascribable to the free coil structures of the single-strand nucleotide sequence. In the absence of K+ , the CD spectra of the S12 solution with different TMPyP4-to-DNA mole ratios (R = 2.3 and 9.2) are presented as the dotted line and the dashed line in Fig. 1(b), respectively. The inset shows the CD intensity plotted against R at characteristic wavelengths of 260 nm, 290 nm and 440 nm. Obviously, mixing TMPyP4 with S12 results in not only the change of the CD signal of S12 (220–325 nm) but also the appearance of the induced CD signal of TMPyP4 (375–500 nm), which is distinctly due to the TMPyP4–S12 inter-molecular interaction. The titration process can be divided into two phases in terms of the R value. (1) 0 < R < 5. The 256-nm peak decreases monotonically and changes into a weak negative one at ∼260 nm, which is accompanied by the occurrence and increase of the 290-nm peak and the strong negative induced CD signal of TMPyP4 at ∼440 nm. These spectral changes indicate the formation of antiparallel G4 structure from the free coil structure of the single-strand S12 directly induced by TMPyP4. (2) 5 < R < 15. The CD signals of both DNA and TMPyP4 almost unchanged in this R range. The turning point of the two phases, i.e. R = 5, can also be taken as the saturated binding number of TMPyP4 molecules to induce the formation of the antiparallel G4 structure. The interaction of TMPyP4 and the single-strand S12 DNA in the absence of K+ was also studied by absorption and fluorescence spectroscopy. The UV–visible absorption spectra of TMPyP4 in Fig. 2(b) show large bathochromic shift (15 nm) and hypochromicity (37%) of the Soret band. The fluorescence spectra of TMPyP4 in Fig. 3(b) present a same trend with the case of the presence of K+ , indicating the strong interaction between TMPyP4 and S12 DNA, which also give us a hint for the formation of G4. In summary, the above results have proved that TMPyP4 serves as a binding molecule that preferentially induces and stabilizes antiparallel S12 DNA G-quadruplex in the absence of K+ .
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Fig. 1. CD spectra of S12 DNA at a concentration of 8.8 × 10−7 M in 10 mM Tris–HCl buffer (pH = 7.2) titrated with TMPyP4 (0–1.67 × 10−5 M) in the presence (a) or absence of K+ (b). Only three representative spectra with the TMPyP4-to-DNA molar ratios (R) are shown, that is, 0 (solid line), 2.3 (dotted line) and 9.2 (dashed line) for each case, and the insets present the intensity of CD signal against R at 260 nm (triangle), 290 nm (circle) and 440 nm (square).
Fig. 2. Absorption spectra of TMPyP4 (4.6 × 10−6 M) in 10 mM Tris–HCl buffer (pH = 7.2) titrated with S12 (0–4.7 × 10−6 M along an arrow) in the presence (a) or absence of K+ (b). Insets show the enlarged Soret bands for clearly seen.
3.3. Interaction of TMPyP4 with the single-strand S6 and S24 DNA in the absence of K+ To gain further insight into the TMPyP4–oligonucleotide interaction and to explore the binding mechanism, we examined the effect of sequence length on the G-quadruplex formation with comparable experiments of a relatively shorter strand S6 and the longer one S24 DNA.
As presented by the CD spectra in Fig. 4, both S6 and S24 DNA were in the form of random coils in the absence of K+ before TMPyP4 was added. Interestingly, after adding TMPyP4, S6 DNA still kept in the random form, whereas S24 DNA transformed into antiparallel G-quadruplex evidenced by an apparent positive CD peak at 290 nm and the corresponding negative induced CD signal of TMPyP4 at 440 nm as reported previously [18]. Probably the mechanism can be analyzed from the different length of the single-strand DNA.
Fig. 3. Fluorescence spectra of TMPyP4 (4.6 × 10−6 M) titrated with S12 (0–4.7 × 10−6 M along an arrow) in the presence (a) or absence of K+ (b) (exc = 400 nm).
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Fig. 4. CD spectra of (a) d(TTAGGG) (S6) and (b) d(TAGGG(TTAGGG)3 T) (S24) without (solid line) and with (dashed line) the addition of TMPyP4 (TMPyP4-to-DNA ratio R = 4) in the absence of K+ .
Scheme 1. Schematic of the antiparallel G4 formation induced by TMPyP4 from the telomeric S24, S12, and S6 DNA.
It is known that S24 DNA has the potential to form intramolecular antiparallel or parallel G4 under proper conditions, S12 has the ability to form hairpin antiparallel G4 or the parallel G4 with two strands, while S6 DNA can only form the intermolecular parallel G4 with four strands, as shown in Scheme 1. In the present study, the formation of antiparallel G4 were observed from S12 and S24 DNA, but not from S6 DNA by the interaction with TMPyP4. Considering the induction behavior of TMPyP4, it is reasonable to assume that the formed G4 should be further stabilized by the binding of TMPyP4. For the cases of S12 and S24 DNA, both the antiparallel G4 structures have the end-loops for the stacking of TMPyP4, which will stabilize G4 further by the aid of the stacked porphyrin. However, the parallel G4 merely have external loops and cannot provide a well space for the accommodation of TMPyP4. Therefore, here we only observed the formation of antiparallel G4 from the S12 and S24 strands by the induction of TMPyP4. For the case of S6 DNA, it cannot form the stable intermolecular four-strand parallel G-quadruplex because this structure cannot provide any loops at the ends of the Gquadruplex for TMPyP4 molecules to interact with. Taking together above analysis, it is reasonable to speculate that the end-loops play crucial roles in stabilizing the G-quadruplex directly induced by TMPyP4. Most recently, Arora and Maiti also revealed an important
effect of the loop regions on the G4–TMPyP4 interaction [17], which is in accordance with our results. In addition, the formation of G4 has also been observed by the direct induction of some organic molecules such as telomestatin [23], anthracene derivatives [24], and tetramethylpyridiniumporphyrazines [25], or some transition metal ions such as Sr2+ [26] and Zn2+ [27] without the involvement of K+ or Na+ . This finding of the induction behavior of TMPyP4 enriches the field of G4 formation, and gives us more information to the binding interactions between porphyrins and G4. 4. Conclusion The interactions between the cationic TMPyP4 and G4 of the human telomeric sequence d(TTAGGG)2 (S12) have been investigated by spectroscopic study. We observed not only the strong binding between TMPyP4 and DNA, but that within a TMPyP4-to-G4 ratio R = 5, TMPyP4 can transform the K+ -induced parallel G4 into the mixed form, and it can directly induce the formation of antiparallel G4 structure without the involvement of K+ . Comparable investigations of TMPyP4 with S6 and S24 DNA suggest that the endloops of the G4 structure play an important role in the induction
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behavior of TMPyP4, which provides useful information of the G4 structure and the binding ability on the drug recognition process. Acknowledgements This work has been supported by the National Natural Science Foundation of China (20772011, 20703067 and 20875009) and the National Basic Research Program of China (973 program, 2007CB914101). References [1] S. Burge, G.N. Pardinson, P. Hazel, A.K. Todd, S. Neidle, Nucleic Acids Res. 34 (2006) 5402–5415. [2] J.K. Davis, Angew. Chem. Int. Ed. 43 (2004) 668–698. [3] T. Simonsson, Biol. Chem. 382 (2001) 621–628. [4] S. Cogoi, L.E. Xodo, Nucleic Acids Res. 34 (2006) 2536–2549. [5] S. Neidle, G.N. Parkinson, Curr. Opin. Struct. Biol. 13 (2003) 275–283. [6] A.M. Zahler, J.R. Williamson, T.R. Cech, D.M. Prescott, Nature 350 (1991) 718–720. [7] R.T. Wheelhouse, D. Sun, H. Han, F.X. Han, L.H. Hurley, J. Am. Chem. Soc. 120 (1998) 3261–3262. [8] I. Haq, J.O. Trent, B.Z. Chowdhry, T.C. Jenkins, J. Am. Chem. Soc. 121 (1999) 1768–1779. [9] H. Arthanari, S. Basu, T.L. Kawano, P.H. Bolton, Nucleic Acids Res. 26 (1998) 3724–3728.
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[10] E. Izbicka, R.T. Wheelhouse, E. Raymond, K.K. Davidson, R.A. Lawrence, D. Sun, B.E. Windle, L.H. Hurley, D.D.V. Hoff, Cancer Res. 59 (1999) 639– 644. [11] H. Han, D.R. Langley, A. Rangan, L.H. Hurley, J. Am. Chem. Soc. 123 (2001) 8902–8913. [12] N.V. Anantha, M. Azam, R.D. Sheardy, Biochemistry 37 (1998) 2709–2714. [13] F.X. Han, R.T. Wheelhouse, L.H. Hurley, J. Am. Chem. Soc. 121 (1999) 3561–3570. [14] G.N. Parkinson, R. Ghosh, S. Neidle, Biochemistry 46 (2007) 2390–2397. [15] C. Wei, G. Jia, J. Yuan, Z. Feng, C. Li, Biochemistry 45 (2006) 6681–6691. [16] H. Mita, T. Ohyama, Y. Tanaka, Y. Yamamoto, Biochemistry 45 (2006) 6765–6772. [17] A. Arora, S. Maiti, J. Phys. Chem. B 112 (2008) 8151–8159. [18] H. Zhang, X. Wang, P. Wang, X. Ai, J. Zhang, Photochem. Photobiol. Sci. 7 (2008) 948–955. [19] C.R. Cantor, M.M. Warshaw, H. Shapiro, Biopolymers 9 (1970) 1059–1077. [20] K. Kalyanasundaram, Inorg. Chem. 23 (1984) 2453–2459. [21] H. Li, O.S. Fedorova, W.R. Trumble, T.R. Fletcher, L. Czuchajowski, Bioconjugate Chem. 8 (1997) 49–56. [22] R. Jasuja, D.M. Jameson, C.K. Nishijo, R.W. Larsen, J. Phys. Chem. B 101 (1997) 1444–1450. [23] M.Y. Kim, M. Gleason-Guzman, E. Izbicka, D. Nishioka, L.H. Hurley, Cancer Res. 63 (2003) 3247–3256. [24] R. Rodriguez, G. Dan Pantos¸, D.P.N. Gonc¸alves, J.K.M. Sanders, S. Balasubramanian, Angew. Chem. Int. Ed. 46 (2007) 5405–5407. [25] D.P.N. Gonc¸alves, R. Rodriguez, S. Balasubramanian, J.K.M. Sanders, Chem. Commun. (2006) 4685–4687. [26] I.M. Pedroso, L.F. Duarte, G. Yanez, A.M. Baker, T.M. Fletcher, Biochem. Biophys. Res. Commun. 358 (2007) 298–303. [27] C. Wei, Q. Tang, C. Li, Biophys. Chem. 132 (2008) 110–113.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin Huijuan Zhang a,∗ , Xiao Xiao a , Peng Wang b , Siping Pang a , Feng Qu a , Xicheng Ai b,∗∗ , Jianping Zhang b a b
School of Life Science and Technology, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 100081, China Department of Chemistry, Renmin University of China, Beijing 100872, China
a r t i c l e
i n f o
Article history: Received 29 November 2008 Received in revised form 28 May 2009 Accepted 7 June 2009 This paper is dedicated to professor Yongzhong Yu on the occasion of his 85th birthday. Keywords: G-quadruplex (G4) TMPyP4 Conformational conversion Spectroscopy
a b s t r a c t The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)2 (S12) have been investigated by means of circular dichroism (CD), UV–visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K+ , and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K+ . Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)3 T) in the absence of K+ show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Guanine-rich sequences exist in many important DNA regions such as telomeres, some oncogenes, immunoglobulin switch regions and recombination hot spots [1–4]. Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG), which can form four-stranded structures called G-quadruplexes (G4) in the presence of K+ or Na+ [5]. The formation of the G4 structures has been shown to decrease the activity of telomerase, which is responsible for elongating telomeres [6]. Therefore, seeking for efficient telomerase inhibitors that are capable of inducing and stabilizing the G4 structure currently attracts great attention in the field of chemotherapy. For the well-matched molecular dimension as well as the strong – stacking and electrostatic interactions, the binding of the cationic porphyrin, meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4), with DNA G4 has been subjected to extensive investigations [7–17]. Many investigations have been focused on the structure of TMPyP4–G4 complex, the binding modes and the binding stoichiometries [14–16], however, the conformational conversion of G4 structure induced by TMPyP4 has not been concerned much in the last decade.
∗ Corresponding author. Tel.: +86 10 6891 2140; fax: +86 10 6891 4607. ∗∗ Corresponding author. Tel.: +86 10 6251 6604; fax: +86 10 6251 6444. E-mail addresses: [email protected] (H. Zhang), [email protected] (X. Ai). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.06.018
In our previous study, we have investigated the interaction between TMPyP4 and G4 formed from the single-strand d(TAGGG(TTAGGG)3 T) (S24) DNA, and firstly found that the conformation of G4 could be transformed by the induction of TMPyP4 [18]. Here we studied the conformational conversion of G4 from d(TTAGGG)2 (S12) DNA induced by TMPyP4 in the presence and absence of K+ . Moreover, comparable experiments of TMPyP4 with S24 and d(TTAGGG) (S6) were made in the absence of K+ , which indicated the important role of the loops for the formation of G4. As there is still no direct evidence for the accurate structure of G4 in vivo, it is of great biological significance to investigate the singlestrand telomeric DNA of S12, S24 and S6 with different length. For the case of S12 DNA, it represents the condition that G4 is formed from two single-strand DNA of two sister chromosomes. For the case of S24 DNA, it represents that G4 is formed from a singlestrand DNA of one chromosome. And for S6, it represents that G4 is formed from four single-strand DNA, which is a simplified model for investigation. 2. Materials and methods 2.1. Sample preparation Oligonucleotides S12, S6, and S24 DNA (PAGE-purified, >99% homogeneous) were purchased from Bioasia Biologic Technology Co. Ltd., and were used without further purification. The solution of DNA was prepared in Tris–HCl buffer (10 mM, pH = 7.2) in the presence of 0 and 10 mM potassium chloride, respectively, and was
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quantitated by the use of UV–visible spectrophotometry at 260 nm with an extinction coefficient of ε260 = 2.29 × 105 M cm−1 per G4 as estimated by using the nearest-neighbor method [19]. TMPyP4 was synthesized following the procedures described in Refs. [20,21]. Fresh solution used in the spectroscopic titration was prepared in the same buffer as those used for DNA, and were quantitated by using ε422 = 2.26 × 105 M−1 cm−1 . To avoid photodegradation, the preparations were kept in the dark before the spectroscopic titration experiments. For the convenience of data analysis, the molar ratio of TMPyP4to-DNA was defined as R in this study. 2.2. Circular dichroism titration The formation of G4 and the induced optical activity of cationic TMPyP4 were monitored by the use of circular dichroism (CD) spectroscopy. The CD spectra were recorded on a CD spectrophotometer (CD6, Jobin-Yvon, Longjumeau, France) and were corrected against the background of buffer. The CD spectra of DNA were examined over 220–325 nm, and those of TMPyP4–DNA complex over 220–500 nm. 2.3. UV–visible absorption and fluorescence titration The binding of TMPyP4 to S12 DNA was monitored by the use of steady-state UV–visible absorption and fluorescence spectroscopies. The final concentration of TMPyP4 was fixed at 4.6 × 10−6 M, to which the annealed DNA solution was added gradually to make a final concentration of S12 in the range of 0–4.7 × 10−6 M (per G4). After incubation at 25 ◦ C for 5 min, the absorption spectrum of each mixture was recorded from 350 nm to 700 nm on a spectrometer (U-3310, Hitachi, Hitachinaka, Japan). Fluorescence spectra of TMPyP4 in the same series of preparation as used in absorption titration were recorded under the photoexcitation at 400 nm on a spectrophotometer (F-2500, Hitachi, Hitachinaka, Japan). 3. Results and discussion 3.1. Conformational conversion of the parallel G4 to the mixed parallel/antiparallel structure from S12 DNA induced by TMPyP4 in the presence of K+ The conformational change of the G4 structure induced by TMPyP4 was examined by means of CD spectroscopy in the presence of K+ (Fig. 1(a)). The solid line in Fig. 1(a) shows the CD spectrum of S12 in Tris–HCl buffer (free from TMPyP4) after annealing treatment, which is characterized by a strong positive peak at 260 nm and a negative one at 239 nm. These features are ascribable to a parallel G4 structure formed from the single-strand S12 DNA in the presence of K+ , which is in accordance with the crystal structure reported by Parkinson et al. recently [14]. With the addition of TMPyP4, the change of the CD spectra of the parallel G4 from S12 are presented in Fig. 1(a) as the dotted line (R = 2.3) and the dashed line (R = 9.2). The inset shows the CD intensity plotted against R at characteristic wavelengths of 260 nm, 290 nm and 440 nm. The addition of TMPyP4 causes apparent decrease of the 260-nm peak and the occurrence of the positive signal at 290 nm until almost keeps constant when R ≈ 5. Meanwhile, a strong negative induced CD peak of porphyrin at 440 nm appears. These changes indicate the transformation of the G4 structure from the parallel type to the mixed parallel/antiparallel one with the binding of TMPyP4 molecules. After R exceeds 5, the CD signal of DNA and TMPyP4 almost keeps constant. The turning point of the titration process that occurs at R ≈ 5, can be regarded as the
saturated binding number of TMPyP4 to induce the G4 structural conversion. The interaction of TMPyP4 and parallel G4 was further investigated by absorption and fluorescence spectroscopy. Fig. 2(a) shows the UV–visible absorption spectra of TMPyP4 (4.6 × 10−6 M) titrated with G4 DNA (0–4.7 × 10−6 M). Significant bathochromic shift (14 nm) and hypochromicity (41%) of the Soret band were observed owing to the strong TMPyP4–G4 interaction. Fig. 3(a) shows the corresponding fluorescence spectra of TMPyP4 titrated with G4 DNA under the excitation wavelength of 400 nm. When free from DNA, TMPyP4 is moderately fluorescent with a fluorescence quantum yield of 0.05. The fluorescence spectrum of whom is characterized by a broad dual-band pattern in the range of 600–800 nm. Upon adding S12, the fluorescence intensity decreases and two peaks at 663 nm and 724 nm become better resolved. The decrease in intensity can be attributed to the quench of singlet excited-state TMPyP4 owing to the electron transfer reaction involving groundstate guanine as the electron donor [22]. Taking together above results, it is shown that TMPyP4 interacts strongly with G4 formed from S12 in the presence of K+ , corresponding to a conformational transition of G4 from the parallel type to the mixed parallel/antiparallel one within a TMPyP4to-DNA ratio of 5. Additionally, to get more information about the G4 conformation, NMR experiment is under study in our laboratory. 3.2. Formation of antiparallel G4 from the single-strand S12 DNA induced by TMPyP4 in the absence of K+ The solid line in Fig. 1(b) shows the CD spectrum of S12 in Tris–HCl buffer (free from both K+ and TMPyP4), which is characterized by a strong positive peak at 256 nm and a weak negative one at 238 nm. These features are ascribable to the free coil structures of the single-strand nucleotide sequence. In the absence of K+ , the CD spectra of the S12 solution with different TMPyP4-to-DNA mole ratios (R = 2.3 and 9.2) are presented as the dotted line and the dashed line in Fig. 1(b), respectively. The inset shows the CD intensity plotted against R at characteristic wavelengths of 260 nm, 290 nm and 440 nm. Obviously, mixing TMPyP4 with S12 results in not only the change of the CD signal of S12 (220–325 nm) but also the appearance of the induced CD signal of TMPyP4 (375–500 nm), which is distinctly due to the TMPyP4–S12 inter-molecular interaction. The titration process can be divided into two phases in terms of the R value. (1) 0 < R < 5. The 256-nm peak decreases monotonically and changes into a weak negative one at ∼260 nm, which is accompanied by the occurrence and increase of the 290-nm peak and the strong negative induced CD signal of TMPyP4 at ∼440 nm. These spectral changes indicate the formation of antiparallel G4 structure from the free coil structure of the single-strand S12 directly induced by TMPyP4. (2) 5 < R < 15. The CD signals of both DNA and TMPyP4 almost unchanged in this R range. The turning point of the two phases, i.e. R = 5, can also be taken as the saturated binding number of TMPyP4 molecules to induce the formation of the antiparallel G4 structure. The interaction of TMPyP4 and the single-strand S12 DNA in the absence of K+ was also studied by absorption and fluorescence spectroscopy. The UV–visible absorption spectra of TMPyP4 in Fig. 2(b) show large bathochromic shift (15 nm) and hypochromicity (37%) of the Soret band. The fluorescence spectra of TMPyP4 in Fig. 3(b) present a same trend with the case of the presence of K+ , indicating the strong interaction between TMPyP4 and S12 DNA, which also give us a hint for the formation of G4. In summary, the above results have proved that TMPyP4 serves as a binding molecule that preferentially induces and stabilizes antiparallel S12 DNA G-quadruplex in the absence of K+ .
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Fig. 1. CD spectra of S12 DNA at a concentration of 8.8 × 10−7 M in 10 mM Tris–HCl buffer (pH = 7.2) titrated with TMPyP4 (0–1.67 × 10−5 M) in the presence (a) or absence of K+ (b). Only three representative spectra with the TMPyP4-to-DNA molar ratios (R) are shown, that is, 0 (solid line), 2.3 (dotted line) and 9.2 (dashed line) for each case, and the insets present the intensity of CD signal against R at 260 nm (triangle), 290 nm (circle) and 440 nm (square).
Fig. 2. Absorption spectra of TMPyP4 (4.6 × 10−6 M) in 10 mM Tris–HCl buffer (pH = 7.2) titrated with S12 (0–4.7 × 10−6 M along an arrow) in the presence (a) or absence of K+ (b). Insets show the enlarged Soret bands for clearly seen.
3.3. Interaction of TMPyP4 with the single-strand S6 and S24 DNA in the absence of K+ To gain further insight into the TMPyP4–oligonucleotide interaction and to explore the binding mechanism, we examined the effect of sequence length on the G-quadruplex formation with comparable experiments of a relatively shorter strand S6 and the longer one S24 DNA.
As presented by the CD spectra in Fig. 4, both S6 and S24 DNA were in the form of random coils in the absence of K+ before TMPyP4 was added. Interestingly, after adding TMPyP4, S6 DNA still kept in the random form, whereas S24 DNA transformed into antiparallel G-quadruplex evidenced by an apparent positive CD peak at 290 nm and the corresponding negative induced CD signal of TMPyP4 at 440 nm as reported previously [18]. Probably the mechanism can be analyzed from the different length of the single-strand DNA.
Fig. 3. Fluorescence spectra of TMPyP4 (4.6 × 10−6 M) titrated with S12 (0–4.7 × 10−6 M along an arrow) in the presence (a) or absence of K+ (b) (exc = 400 nm).
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Fig. 4. CD spectra of (a) d(TTAGGG) (S6) and (b) d(TAGGG(TTAGGG)3 T) (S24) without (solid line) and with (dashed line) the addition of TMPyP4 (TMPyP4-to-DNA ratio R = 4) in the absence of K+ .
Scheme 1. Schematic of the antiparallel G4 formation induced by TMPyP4 from the telomeric S24, S12, and S6 DNA.
It is known that S24 DNA has the potential to form intramolecular antiparallel or parallel G4 under proper conditions, S12 has the ability to form hairpin antiparallel G4 or the parallel G4 with two strands, while S6 DNA can only form the intermolecular parallel G4 with four strands, as shown in Scheme 1. In the present study, the formation of antiparallel G4 were observed from S12 and S24 DNA, but not from S6 DNA by the interaction with TMPyP4. Considering the induction behavior of TMPyP4, it is reasonable to assume that the formed G4 should be further stabilized by the binding of TMPyP4. For the cases of S12 and S24 DNA, both the antiparallel G4 structures have the end-loops for the stacking of TMPyP4, which will stabilize G4 further by the aid of the stacked porphyrin. However, the parallel G4 merely have external loops and cannot provide a well space for the accommodation of TMPyP4. Therefore, here we only observed the formation of antiparallel G4 from the S12 and S24 strands by the induction of TMPyP4. For the case of S6 DNA, it cannot form the stable intermolecular four-strand parallel G-quadruplex because this structure cannot provide any loops at the ends of the Gquadruplex for TMPyP4 molecules to interact with. Taking together above analysis, it is reasonable to speculate that the end-loops play crucial roles in stabilizing the G-quadruplex directly induced by TMPyP4. Most recently, Arora and Maiti also revealed an important
effect of the loop regions on the G4–TMPyP4 interaction [17], which is in accordance with our results. In addition, the formation of G4 has also been observed by the direct induction of some organic molecules such as telomestatin [23], anthracene derivatives [24], and tetramethylpyridiniumporphyrazines [25], or some transition metal ions such as Sr2+ [26] and Zn2+ [27] without the involvement of K+ or Na+ . This finding of the induction behavior of TMPyP4 enriches the field of G4 formation, and gives us more information to the binding interactions between porphyrins and G4. 4. Conclusion The interactions between the cationic TMPyP4 and G4 of the human telomeric sequence d(TTAGGG)2 (S12) have been investigated by spectroscopic study. We observed not only the strong binding between TMPyP4 and DNA, but that within a TMPyP4-to-G4 ratio R = 5, TMPyP4 can transform the K+ -induced parallel G4 into the mixed form, and it can directly induce the formation of antiparallel G4 structure without the involvement of K+ . Comparable investigations of TMPyP4 with S6 and S24 DNA suggest that the endloops of the G4 structure play an important role in the induction
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