Studies on the DNA binding and cleavage activity of a synthesized polyamide containing dipeptide Ser-His

Transcription

1 Science in China Series B: Chemistry 2007 SCIENCE IN CHINA PRESS Springer Studies on the DNA binding and cleavage activity of a synthesized polyamide containing dipeptide Ser-His ZHANG Zhen, NIU MingYu, JU Yong & ZHAO YuFen Key Laboratory of Bioorganic Phosphorous Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing , China The DNA binding and cleavage activity of a synthetic polyamide containing dipeptide Ser-His has been investigated by spectroscopic techniques, such as electronic absorbance and circular dichroism (CD) spectropolarimetry, as well as gel electrophoresis. The results show that the molecule has a strong interaction with DNA and can improve DNA-cleavage ability about 100 fold as compared with single dipeptide Ser-His. polyamide, DNA binding affinity, spectroscopy, DNA cleavage In recent decades considerable attention has been focused on new DNA binding and modifying agents, which range from natural products to wholly synthetic designs. These small molecules serve as probes for DNA structure in solution, as agents for mediation of strand scission of duplex DNA and as potential chemotherapeutic agents [1-4]. Results of our preceding experiments show that dipeptide Ser-His has the ability to interact with multiple classes of biological molecules over wide ranges of physical and chemical conditions. To the best of our knowledge, dipeptide Ser-His is the shortest peptide ever reported to have multiple cleavage activities [5]. However, these results illustrate that the DNA cleavage activity of dipeptide Ser-His is relatively low and not sequence-specific. Currently, polyamides containing N-methylpyrrole and N-methylimidazole have been extensively investigated by synthetic and biological chemists, due to their ability to recognize and bind to the minor groove of DNA [6]. Therefore, with an attempt to improve the cleavage ability and sequence-specificity of DNA, a new polyamide 1 containing dipeptide Ser-His was synthesized, whose structure features a dipeptide L-Ser-L-His subunit linked to a polyamide moiety consisting of three N-methylpyrroles through an alkyl spacer (Figure 1). We report herein the DNA binding and cleavage activity with this molecule by electronic absorbance titration, thermal denaturation, and circular dichroism (CD) spectropolarimetry, as well as gel electrophoresis. These investigations might provide fundamental information for further elucidating the curing mechanism and rationally designing more efficient new therapeutic agents. Figure 1 Chemical structure of polyamide 1. Received May 27, 2006; accepted May 22, 2007 doi: /s z Corresponding author ( yfzhao@mail.tsinghua.edu.cn) Supported by the National Natural Science Foundation of China (Grant No ) Sci China Ser B-Chem Dec vol. 50 no

2 1 Methods and materials 1.1 Chemicals and buffer conditions Polyamide 1 was synthesized according to the published procedure [7] and was confirmed by 1 H NMR, IR, ESI-MS and HR-MS. Stocking solution of 1 was prepared in H 2 O/CH 3 OH, 3:1 (volume ratio) and kept by freezing at 20. All chemicals and solvents were of analytical grade or higher and were used without further purification. Deionized water was used throughout. All of the spectroscopic experiments were conducted in 10 mmol/l Tris-HCl (ph 7.0), 20 mmol/l NaCl. For DNA cleavage experiments, 40 mmol/l Britton-Robinson buffer (B-R buffer, equal amounts of phosphate, borate, and acetate) was used, containing 5 mmol/l NaCl, ph DNA samples Calf thymus DNA (CT DNA), poly(da-dt) and poly(dg-dc) were purchased from Sigma (USA). Supercoiled plasmid pbr322 DNA was obtained from Sino-American Biothechnology Co. (China). They were used as received. These sodium salts were stored at below 4. Stocking solutions of CT DNA, poly(da-dt) and poly(dg-dc) were prepared in 10 mmol/l Tris-HCl (ph 7.0), 20 mmol/l NaCl. Purity of the DNA samples was checked by monitoring the absorption spectrum and the ratio of the absorbance at 260 to 280 nm. DNA concentrations per nucleotide were determined by absorption spectroscopy, using the following molar extinction coefficients at the indicated wavelengths: CT DNA, 6600 mol 1 L cm 1, 260 nm; poly(da-dt), 6600 mol 1 L cm 1, 260 nm; poly(dg-dc), 8400 mol 1 L cm 1, 254 nm [8,9]. 1.3 Absorbance titration studies All absorption spectra were acquired at 25 on an Ultrospec 4000 UV/Visible spectrophotomer equipped with a thermoelectrically controlled cell holder. The absorbance titrations were performed as reported in literature [10]. Typically, the measurements were carried out by keeping the concentration of polyamide 1 constant while varying the concentration of CT DNA. The absorbance at 297 nm was recorded after each addition of CT DNA. The absorption data were analyzed to evaluate the binding constant K b, which can be determined from eq. (1), [DNA]/(ε A ε F) = [DNA]/(ε B ε F) + 1/K b (ε B ε F), (1) where ε A corresponds to A obsd /[1], and ε F and ε B are the extinction coefficients for the free polyamide 1 and polyamide 1 in the fully bound form, respectively. 1.4 Measurement of denaturation temperature Absorbance versus temperature profiles were measured at 260 nm using a computer-interfaced Ultrospec 4000 UV/Visible spectrophotomer equipped with a thermoelectrically controlled cell holder. The heating rate in all experiments was 0.5 /min. For each optically detected transition, the melting temperature (T m ) was determined as described in literature [11]. 1.5 CD titration studies CD spectra were recorded by using a Jasco J715 spectropolarimeter attached to a Julabo water circulator for variable temperature. Initially, DNA was placed in the cell and the spectrum of the DNA alone was reported. Aliquots of ligand were then added and each of the spectra was reported. All measurements were made at 25. The spectra collected were from 400 to 220 nm with a scan speed of 200 nm/min. Two scans were accumulated and automatically averaged by the computer. 1.6 DNA cleavage experiments For gel electrophoresis experiments, supercoiled pbr322 DNA (400 ng) was treated with varying concentrations of 1 (0-400 μmol/l) in 40 mmol/l B-R buffer (ph 6.0, containing 5 mmol/l NaCl) to a total volume of 10 μl. The solutions were incubated for 48 h at 37, and then quenched by freezing at 20. The samples were analyzed by electrophoresis for 1.5 h at 50 V on a 1% agarose gel in Tris-acetic acid-edta buffer. The gel was stained with 0.5 mg/l EB and photographed under UV light. 2 Results and discussion 2.1 Absorbance titration studies The electronic absorption spectra of 1 (Figure 2(a)) in the presence of increasing amounts of CT DNA showed strong decreases in the peak intensities (~50% hypochromicity). A modest bathochromic shift (ca. 14 nm shift of the maximum) was observed. The hypochromism in the electronic absorption spectra is thought to be due to a strong interaction between 1 and DNA double strands. A continuous decrease in the intensity of 1 absorption was followed by saturation at high concentrations of DNA (inset in Figure 2(a)). The half-reciprocal plot of the absorption titration data according to eq. (1) ZHANG Zhen et al. Sci China Ser B-Chem Dec vol. 50 no

3 EB started to increase gradually. This phenomenon indicated that polyamide 1 could interact with DNA and dismantle the intercalation, and therefore EB was released from DNA. However, the same phenomenon could not be observed from Ser-His when the same performance was done, which clearly showed that interaction between Ser-His and DNA was quite weak. Figure 2 (a) Absorption spectral changes of 1 upon the addition of CT DNA. [1]= 20 μmol L 1, [DNA]= μmol L 1 in 10 mmol L 1 Tris-HCl (ph 7.0) with 20 mmol L 1 NaCl at 25. Inset: absorbance changes of 1 at 297 nm upon the addition of CT DNA. (b) A half-reciprocal plot of 1 binding with CT DNA as determined from the absorbance titration. gave a linear plot and resulted in an intrinsic binding constant (K b ) of (2.0 ± 0.2) 10 4 mol 1 L (Figure 2(b)). Due to the absence of absorption in the nm wavelength region for dipeptide Ser-His molecule, we chose EB as a probe to compare the interaction intensity of polyamide 1 and Ser-His with DNA indirectly. Control experiments were performed in the presence of EB and CT DNA with different concentrations of 1 and Ser-His (Figure 3), respectively. After the addition of CT DNA, the absorption intensity of EB decreased, which resulted from the intercalation of the EB planar phenanthridinium ring into adjacent base pairs of DNA [12]. When polyamide 1 was continuously added into the DNA/EB solution, the absorption intensity of Figure 3 UV-visible absorption spectra in 10 mmol L 1 Tris-HCl (ph 7.0) with 20 mmol L 1 NaCl at 25 in the presence of 20 μmol L 1 EB and 80 μmol L 1 CT DNA with different concentrations of: (a) polyamide 1, 3-6: 20, 40, 60, 80 μmol L 1 ; (b) Ser-His, 3-6: 20, 40, 60, 80 μmol L 1. For both (a) and (b): 1, 20 μmol L 1 EB; 2, 20 μmol L 1 EB + 80 μmol L 1 CT DNA. 2.2 DNA melting studies The denaturation of DNA from double-stranded to single-stranded is reflected by the absorption hyperchromism around 260 nm. Thus, the helix to coil transition 808 ZHANG Zhen et al. Sci China Ser B-Chem Dec vol. 50 no

4 temperature can be determined by monitoring the absorbance of DNA bases at 260 nm as a function of temperature. The binding of small molecules to the double-stranded DNA usually results in the stabilization of the duplex structure to some extent depending on the strength of their interactions. The binding should lead to an increase in the melting temperature of DNA as compared with DNA alone. The DNA melting curves in Figure 4 showed that the melting temperature of free double helical CT DNA was 76.9, and the binding of Ser-His was the same while the binding of polyamide 1 showed a value of ΔT m 2.0 increment. Figure 4 UV melting of DNA thermal denaturation in 10 mmol L 1 Tris-HCl, 20 mmol L 1 NaCl, ph 7.0., CT DNA (150 μmol L 1 );, CT DNA (150 μmol L 1 ) with Ser-His at [DNA]:[Ser-His]= 2:1; Δ, CT DNA (150 μmol L 1 ) with 1 at [DNA] : [1]= 2: CD titration studies Although CD spectra changes cannot be interpreted on a quantitative theoretical basis, CD is still one of the most powerful techniques for investigating the solution structure of DNA and its conformational modifications produced by ligand binding. The CD signature for B-form DNA is a positive band centered at 275 nm and a negative band at 240 nm, with the zero point around 258 nm. We first investigated the CD spectra of both polyamide 1 and Ser-His in the nm wavelength region to determine whether the ligands would result in disturbance. For polyamide 1, there was no CD signal. Furthermore, for Ser-His, there was a positive band at about 213 nm (data not shown). The interactions of both 1 and Ser-His with CT DNA, poly(da-dt) and poly(dgdc) were monitored by CD measurements. The representative CD spectra are shown in Figure 5. Small changes in the CD spectra were observed upon titration of Ser-His, thereby suggesting that the dipeptide has week interactions with the DNAs. However, titration of polyamide 1 to the DNAs showed much stronger signature by the appearance of DNA-induced ligand CD bands at about nm, presumably due to the UV absorption π to π * transition of the molecule in the ligand DNA complex [13]. The appearance of a DNAinduced ligand CD band is clear evidence of the interactions of the molecule with the DNAs, because neither the free DNAs nor the free polyamide 1 exhibited CD signals in the nm wavelength region. The maximum absorption signals were up to an ultimate value with the addition of surplus ligands. Titration of 1 to CT DNA produced a strong positive band at 328 nm (6.5 mdeg, r = 0.36, where r values are defined as the input ration of the moles of ligand to DNA base pairs) with the isoelliptic points at 257 and 290 nm. Addition of 1 to poly(da-dt) produced a positive band at 325 nm (3.0 mdeg, r = 3.58) with the isoelliptic points at 232, 256 and 262 nm. However, titration of 1 to poly(dg-dc) gave rise to a negative band at 328 nm ( 1.7 mdeg, r = 2.48) sharing the two isoelliptic points at 242 and 302 nm. In the above CD experiments, the presence of the negative and positive bands of CT DNA at ~245 and ~270 nm, respectively, suggests that the conformation of CT DNA in the ligand DNA complex remained in the B-form. However, for both poly(da-dt) and poly(dgdc), this conjugate caused disappearance of the negative band at ~245 nm. 2.4 DNA cleavage experiments The polyamide is capable of cleaving double-stranded DNA at physiological ph and temperature. When plasmid pbr322 DNA was incubated with 1, the DNA degraded from form Ι (supercoiled) to form ΙΙ (nicked circular), and then to form ΙΙΙ (linear). Figure 6 shows agarose gel electrophoresis patterns for the cleavage of plasmid pbr322 DNA after being treated with 1 at ph 6.0 (40 mmol/l B-R buffer, 5 mmol/l NaCl) and 37 for 48 h. The initial concentration of DNA was set at 40 mg/l, and the concentration of 1 was varied from 0 to ZHANG Zhen et al. Sci China Ser B-Chem Dec vol. 50 no

5 400 μmol/l. The conversions of form Ι to form ΙΙ and to form ΙΙΙ were observed with the increasing concentration of 1, and form ΙΙΙ began to appear in the presence of 120 μmol/l of 1 (lane 6 in Figure 6). Form Ι was barely observable in lane 8 (200 μmol/l of 1). Still higher concentrations (>1.0 mmol/l of 1) led to precipitation of the plasmid DNA. According to our proceeding experiments, 20 mmol/l Ser-His is needed when form I is barely observable under the same conditions[5]. Compared to single dipeptide Ser-His molecule, this new synthetic polyamide 1 enhanced DNA-cleavage ability about 100 fold. Figure 5 (a) (c): CD titration of polyamide 1 into (a) CT DNA (105.0 μmol L 1), r = ; (b) poly(da-dt) (21.8 μmol L 1), r = ; (c) poly(dg-dc) (31.5 μmol L 1), r = ; (a ) (c ): CD titration of Ser-His into (a) CT DNA (150.0 μmol L 1), r = ; (b) poly(da-dt) (28.4 μmol L 1), r = ; (c) poly(dg-dc) (44.0 μmol L 1), r = The r values are defined as the input ration of the moles of ligand to DNA base pairs. 810 ZHANG Zhen et al. Sci China Ser B-Chem Dec vol. 50 no

6 1 could improve DNA-cleavage ability greatly. 3 Conclusion Figure 6 Agarose gel electrophoresis patterns for the cleavage of pbr322 DNA with various concentrations of 1 in 40 mmol L 1 B-R buffer, containing 5 mmol L 1 NaCl, ph 6.0 at 37 for 48 h. Lane 1, DNA control; lanes 2-9, cleavage patterns in the presence of 20, 60, 80, 100, 120, 150, 200, 400 μmol L 1 of 1, respectively. Data from all of the spectroscopic experiments clearly showed that polyamide 1 had much stronger binding affinity than Ser-His due to the introduction of three N-methylpyrroles, which functioned like an anchor and increased the interaction between the small molecules and DNA. Therefore, it is no doubt that polyamide Within this frame the interaction of the new synthetic polyamide with DNA was analyzed in vitro through a variety of classical physiochemical techniques, as well as gel electrophoresis. The results illustrate that polyamide 1 has a strong interaction with DNA and can improve DNA-cleavage ability about 100 fold compared to single dipeptide Ser-His. These investigations may usefully improve the understanding of the mechanics and the activity of both naturally and artificially occurring molecules, thus giving further signposts in the ongoing research into nucleic acids. 1 Dervan P B. Molecular recognition of DNA by small molecules. Bioorg & Med Chem, 2001, 9(9): Rucker V C, Foister S, Melander C, Dervan P B. Sequence specific fluorescence detection of double strand. J Am Chem Soc, 2003, 125(5): Zimmer C, Wahnert U. Nonintercalating DNA-binding ligands: Specificity of the interaction and their use as tools in biophysical, biochemical and biological investigation of the genetic material. Prog Biophys Mol Biol, 1986, 47(1): Burrows C J, Rokita S E. Recognition of guanine structure in nucleic acids by nickel complexes. Acc Chem Res, 1994, 27(10): Li Y S, Zhao F, Hatfield S, Wan R, Zhu Q, Li X H, McMills M, Ma Y, Li J, Brown K L, He C, Liu F, Chen X Z. Dipeptide seryl-histidine and related oligopeptides cleave DNA, protein, and a carboxyl ester. Bioorg Med Chem, 2000, 8(12): Wemmer D E, Dervan P B. Targeting the minor groove of DNA. Curr Opin Struct Biol, 1997, 7(3): Ye Y, Niu M Y, Yin Q, Cao L F, Zhao Y F. A synthetic route to N-methylpyrrole containing polyamide/peptide conjugate. J Chem Res S, 2005, (4): Pyle A M, Rehmann J P, Meshoyrer R, Kumar C V, Turro N J, Barton J K. Mixed-ligand complexes of ruthenium (II): Factors governing binding to DNA. J Am Chem Soc, 1989, 111(8): Wolfe A, Shimer G H, Jr Mechan T. Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry, 1987, 26(20): Zhong W Y, Yu J, Huang W L, Ni K Y, Liang Y Q. Spectroscopic studies of interaction of chlorobenzylidine with DNA. Biopolymers, 2001, 62(6): Gore M G. Spectrophotometry and Spectrofluorimetry. New York: Oxford University Press, Reinhardt C G, Krugh T R. A comparative study of ethidium bromide complexes with dinucleotides and DNA: Direct evidence for intercalation and nucleic acid sequence preferencest. Biochemistry, 1978, 17(23): Lee M, Rhodes A L, Wyatt M D, Forrow S, Hartley J A. GC base sequence recognition by oligoimidazolecarboxamide and C-terminusmodified analogs of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron (II) footprinting studies. Biochemistry, 1993, 32(16): ZHANG Zhen et al. Sci China Ser B-Chem Dec vol. 50 no