Metal-Free DNA Linearized Nuclease Based on PASP–Polyamine

Article pubs.acs.org/bc

Metal-Free DNA Linearized Nuclease Based on PASP−Polyamine Conjugates Chao Li,† Fangfang Zhao,† Yunan Huang,† Xueyuan Liu,† Yan Liu,§ Renzhong Qiao,*,†,‡ and Yufen Zhao§ †

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, 100083, China § Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡

S Supporting Information *

ABSTRACT: Genome manipulation controlled by small metal complexes has attracted extensive interest because of their potential application in the fields of molecular biotechnology and drug development. However, their medicinal application is still limited due to the distinct toxicity of the free radicals generated by partial metal complexes based on oxidative cleaving processes. Thus, it is still a challenge for us to use metal free agent to cleave DNA. In this work, we showed that a family of polyamine-grafted PASP (poly(aspartic acid)) conjugates is able to rapidly induce DNA cleavage in the absence of metal ions, and obtain a high-yield linearization product via a hydrolytic path. From the results of detailed control experiments, it was revealed that the formation of polyamine cation/phosphate anion pair and free ungrafted nucleophilic groups would be the key factors to improve DNA linearization. Constructing polyamine conjugates based on short peptide such as polyamine-grafted PASP, as achieved here, could provide an attractive strategy for developing mild and efficient artificial nucleases as well as researching catalytic mechanisms on DNA chemistry.

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INTRODUCTION Artificial nucleases have attracted significant interest due to their abilities in accelerating DNA cleavage, which makes possible the DNA manipulations that are essential in biotechnology, medicine, and other fields.1,2 Ligand-based hydrolytic DNA cleavage remains a challenge, as it is wellknown that the half-life of DNA by spontaneous hydrolysis under physiological conditions is estimated as thousands to billions of years, which is primarily due to the repulsion between the negatively charged backbone and potential nucleophiles.3 In the past few years, several research groups have been working on the design and evaluation of efficient DNA cleavage agents that are hydrolytic or oxidative in manner. Up to now, almost all of these obtained artificial agents need specific metal ions as cofactors for sequence-specific cleavage.4,5 In spite of the successful results involving artificial metallonucleases, studies devoted to metal-free organic molecules as nucleic acid cleavage agents are not frequent.6−8 Actually, the cleavage of DNA by metal-free compounds can be considered safer in the development of biotechnology, especially in gene therapy, since the absence of a redox active metal ion has the advantage of avoiding complications such as metal dissociation and uncontrolled redox chemistry reactions.9,10 Recently, metal-free DNA cleaving reagents have been pushed forward by some designs of efficient small organic molecules including guanidinium derivatives,11,12 cyclodextrin derivatives,13 and macrocyclic polyamine.8 In addition, seryl© 2012 American Chemical Society

histidine as the shortest functional dipeptide reported in our previous works14−17 exhibits high DNA cleavage activity, because the side chain hydroxyl of serine often serves as a nucleophile, while the side chain imidazole of histidine can serve as a proton donating or accepting group in enzymes. An important property of seryl-histidine is that it can induce efficient DNA hydrolysis in the absence of any metals. To explore its possible mechanism further in this work, we attempted to construct a longer peptide molecule than our previous work to evaluate whether it can maintain cleavage activity like dipeptide or superior properties. In the current work, we use PASP as a simple peptide to establish a new short peptide-based artificial nuclease, grafting polyamine (cyclen, tacn, and cyclam) to carboxyl groups of PASP (Scheme 1), and expect to have higher DNA binding affinity, since the excessive anionic carboxyl group in PASP will show electrostatic repulsion with the anionic phosphate backbone of DNA. The cleavage of DNA was investigated by agarose gel electrophoresis and atomic force microscopy (AFM). The interactions between DNA and PASP-polyamine were further studied by circular dichroism (CD) and fluorescence quenching. To further explore a possible mechanism and structure activity relationship of DNA cleavage induced by conjugates, a series of Received: March 27, 2012 Revised: June 13, 2012 Published: August 8, 2012 1832

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Fluorescence Quenching Assay. Fluorescence spectra were recorded on a Hitachi model F-4500 spectrofluorimeter, with excitation and emission band bass: 10 nm (λex = 520, λem = 620 nm). A 10 mg·mL−1 solution of PASP−polyamine was titrated into the DNA-EB solution (60 μg·mL−1 calf thymus DNA solution including 0.1 mg·mL−1 EB) at pH 7.4 10 mM Tris−HCl buffer with 10 mM NaCl. Apparent binding constants (Kapp) of the ligands with CT-DNA (calf thymus DNA, 60 μg·mL−1) were estimated and compared by measuring the loss of EtBr fluorescence as a function of added ligand. The Kapp values were calculated from: KEtBr[EtBr] = Kapp[Q], where [EtBr] and KEtBr are the concentration and binding constant of EtBr, respectively, and [Q] is the concentration of quencher at 50% of maximal EtBr fluorescence. The binding constant of EtBr was taken to be 1 × 107. Circular Dichroism (CD). All experiments were performed with a continuous flow of nitrogen purging the Jasco-810 spectropolarimeter with a path length cell of 1 cm at room temperature. A 10 mg·mL−1 solution of all samples was titrated into the DNA solutions (at pH 7.4, 10 mM Tris−HCl buffer with 10 mM NaCl) with a final concentration of 1.1 × 10−4 M. The standard scan parameters for all experiments used a wavelength range from 400 to 220 nm. Sensitivity was set at 100 mdeg and scan speed of 200 nm per minute. Three scans were made and the average value of them was calculated. AFM Assay. Atomic force microscope (AFM) imaging was performed in tapping mode on a Digital Instruments multimode NanoScope III having a maximal lateral range of approximately 5 μm. All images were analyzed by tapping in air. High-quality mica sheets (FluorMica) were cut with scissors into squares (1 cm × 1 cm) and attached with superglue to 15 mm round stainless steel sample disks (Ted Pella). Before each use, the mica was freshly cleaved by pulling off the top sheets with tape and then covered with 10 μL of autoclaved AFM buffer (10 mM Tris pH 7.5, 1 mM EDTA, 5 mM MgCl2). The surface is precoated with Mg2+ to allow negatively charged DNA to bind. After 5 min, the buffer was rinsed thoroughly with 0.5 mL of distilled water, and the mica was briefly dried under a stream of N2 (g). The DNA sample was diluted with AFM buffer to 2.5 ng μL−1, and then, 10 μL diluted sample was dropwise added to mica surface. After 5 min, the buffer was rinsed thoroughly with 0.5 mL of distilled water, and the mica was briefly dried under a stream of N2 (g).

Scheme 1. Structures of PASP−Polyamine and Control Conjugates

ingenious control compounds were prepared and evaluated in comparison with the title compounds, as shown in Scheme 1.

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EXPERIMENTAL PROCEDURES General Information. MS (ESI) mass spectral data were recorded on a Finnigan LCQDECA mass spectrometer. 1H NMR and 13C NMR spectra were measured on a Bruker AV600 spectrometer, and chemical shifts in ppm are reported relative to internal Me4Si (CDCl3). All other chemicals and reagents were obtained commercially and used without further purification. Electrophoresis grade agarose and plasmid DNA (pUC18) were purchased from Promega Corporation. The molecular weight (MW) of the polymeric conjugate was measured by Maldi-Tof (Bruker Autoflex). Metal contents in deionized water and buffer solutions were measured by an Agilent 7500a inductively coupled plasma mass spectrometer (Agilent, Japan) system. Maldi-Tof MS. Maldi-Tof MS was carried out on a Bruker Autoflex operating in reflected mode. 2-(4-Hydroxyphenylazo)benzoic acid (HABA) was used as matrix, and NaCl or KCl was used as cationizing agent. Samples were dissolved in MeOH/ H2O (1:1) at a concentration of 1.0 μg·mL−1. HABA was dissolved in dioxane at a concentration of 0.05 M. Sample (20 μL) and matrix (80 μL) solutions were mixed, and then, 80 μL of 0.02 M NaCl or KCl was added. Finally, 1 μL of the resulting mixture was placed on the Maldi plate. Gel Electrophoresis Assay. Electrophoresis experiments were performed with plasmid DNA (pUC18). In a typical experiment, supercoiled pUC18 DNA (5 μL, 0.05 μg·μL−1) in Tris−HCl buffer (40 mM, pH 7.4) was treated with different concentration catalyst, followed by dilution with the Tris−HCl buffer to a total volume of 80 μL. The samples were then incubated at different temperature and time, and loaded on a 1% agarose gel containing 1.0 μg·mL−1 ethidium bromide (EB). Electrophoresis apparatus consisted of a Biomeans Stack IIElectrophoresis system, PPSV-010. Electrophoresis was carried out at 85 V for 1 h in TAE buffer, and bands were visualized by UV light and photographed, recorded on an Olympus GrabIT2.0 Annotating Image Computer System.

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RESULTS AND DISCUSSION Synthesis and Characterization of PASP−Polyamine and Their Control Derivatives. In this study, polyamine including tacn, cyclen, and cyclam is introduced to the carboxyl groups of PASP with a flexible linker; this process is realized by the typical hydrolysis reaction of poly(succinimide) (PSI) and polyamine containing the NH2-terminal group. Initially, PSI as starting material for all compounds is prepared according to relative literature.18,19 The remaining succinimide units after the grafting reaction are further hydrolyzed in the presence of NaOH, propylamine, ethanolamine, and N,N-dimethyl propylene diamine, thereby giving the title compounds, PASP-cyclenpropyl, PASP-cyclen-hydroxyethyl, and PASP-Dp, respectively. The route of synthesis and the NMR data are shown in SI Scheme S1 and Figures S1−S5, respectively. To provide a credible control assay and present a convincing mechanism, PASP−polyamines with a similar degree of 1833

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production of linear DNA is observed within 30 min for PASP−cyclen. The profile for PASP−cyclen mediated reaction displays approximately pseudo-first-order kinetic behavior, with kobs ≈ 0.585 h−1 and R2 = 0.98. The data after 6 h are omitted from Figure 1B since the DNA has already begun to smear as a result of multiple cleavage events. Subsequently, a standard statistical test for determining linearization with random or nonrandom DNA cleavage is applied. According to the Freifelder-Trumbo relationship,20,21 the ratio of n1/n2 is just 1.24 ± 0.36 in our experiments, suggesting a nonrandom cleavage path by PASP−cyclen to efficiently form linear DNA. The cleavage behaviors of PASP−tacn and PASP−cyclam under the same conditions are similar to that of PASP−cyclen (SI Figure S7). A point worth emphasizing is that no extra metal ion is involved in the whole cleavage processes. The content of residual metals in cleavage reactions including Cu, Zn, Fe, and Co is measured by inductively coupled plasma-mass spectroscopy (ICP-MS). The data are listed in SI Table S1, indicating the content that is not enough to attack DNA with any pathways based on control trials. Visual Presentation of DNA Linearization and Ligation−AFM Images. To get visible evidence about DNA cleavage mode and mechanism, AFM studies were performed. Without any treatment, the free DNA exists as supercoiled, and a little loose nicked forms (Figure 2A). After the addition of 38.2 μg·mL−1 or 79.6 μg·mL−1 PASP−cyclen, the mixtures are incubated in Tris−HCl buffer (pH 7.4) at 37 °C for 3 h, and the original supercoiled and nicked DNA turn into linear DNA (Figure 2B) or smaller fragments (Figure 2C), respectively. Subsequently, the extracted linear DNA generated from the reaction above are treated with T4 DNA ligase, and then, longer linear DNA are observed clearly in the AFM images (Figure 2D). This phenomenon shows DNA linkage in the presence of T4 DNA ligase, and it also proves that terminal groups of these fragments provided 3′-hydroxyls and 5′phosphates, since DNA ligase requires them for ligation. The generation of these terminal groups is consistent with the

polyamine substitute, such as PASP−cyclen (53.2%), PASP− tacn (48.9%), and PASP−cyclam (47.3%), are selected as candidates. The method of synthesis of control compounds endows them with the same degree of cyclen substitutes. The degree of substituted polyamines is calculated through the NMR data (see Supporting Information). The molecular weight range and TGA data of polyamine are given in SI Figure S6 and Table S2, respectively. Electrophoresis Results of PASP−Cyclen Induced DNA Linearization. PASP−polyamine is found to mediate the rapid degradation of supercoiled (form I) plasmid DNA under mild conditions (pH 7.4, 37 °C). The DNA cleavage chemistry of PASP−polyamine is evaluated by concentration- and timedependent assay. Importantly, linear (form III) DNA form prior to nicked (form II) under both time and concentration courses, suggesting excellent activity of DNA double-strand cleavage. In the case of PASP−cyclen, a mass of well-defined linear DNA is observed when the concentration is just 9.6 μg·mL−1. With the increase of the agent up to 38.2 μg·mL−1, plasmid DNA is almost linearized at 37 °C for 3 h. Linear DNA is gradually degraded into smears of progressively smaller fragments when the excessive PASP−cyclen is added into the reaction (Figure 1A). In time-dependent assay, quantitative

Figure 1. (A) Concentration dependence of pUC18 DNA (0.05 μg·μL−1) cleavage by PASP−cyclen in 40 mM pH 7.4 Tris−HCl buffer for 3 h at 37 °C. Lanes 1−11: 0, 2.4, 9.6, 16.8, 23.8, 31.0, 38.2, 45.4, 52.6, 79.6, and 86.8 μg·mL−1, respectively. (B) Time dependence of pUC18 DNA cleavage in the presence of 38.2 μg·mL−1 PASP−cyclen in 40 mM pH 7.4 Tris−HCl buffer at 37 °C. Lane 1, control; lanes 2− 14: 0, 0.5, 1, 2, 4, 6, 8, 12, 16, 20, 24, 28, and 32 h. S: supercoiled DNA, N: nicked DNA, L: linear DNA.

Figure 2. AFM images of pUC18 DNA molecules for different forms on mica in trap mode. (A) Supercoiled DNA without any treatment; (B) and (C) treated with PASP−cyclen (38.2 μg·mL−1 and 79.6 μg·mL−1) in Tris−HCl (pH 7.4) for 3 h at 37 °C, respectively; (D) recovered DNA with T4 DNA ligase for 12 h at 4 °C, respectively. Scale bar is 500 nm. 1834

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Figure 3. Fluorescence quenching by (A) PASP/CT-DNA and (B) PASP−cyclen/CT-DNA at pH 7.4 (10 mM Tris−HCl buffer with 10 mM NaCl) with quencher concentration of 0−90.0 μg·mL−1 (from top to bottom). (C) Plots of fluorescence quenching of CT-DNA by PASP (■) and PASP− cyclen (●).

Figure 4. CD spectra of (A) DNA/control compounds (dash, DNA only; blue, DNA/PASP; green, DNA/cyclen-NH2) and (B) DNA/PASP− polyamine with different ligands (dash, DNA only; red, DNA/PASP−cyclen; purple, DNA/PASP−tacn; desert tan, DNA/PASP−cyclam).

cleavage of phosphodiesters, and would be a characteristic of DNA hydrolysis cleavage. Interaction between PASP−Polyamine and DNA. A competitive displacement assay using PASP and PASP− polyamine as quencher can characterize the affinity to DNA. To better demonstrate whether the PASP−cyclen conjugate possesses a better affinity to DNA compared with unmodified PASP, both fluorescence changes are recorded as shown in Figure 3. The fluorescence of PASP/DNA decreases by up to 17.8% with PASP concentration of 90.0 μg·mL−1 (Figure 3A). It can be seen that, with the increase in the amount of PASP, the intensity of fluorescence decreases, indicating that EB is replaced by the added PASP; that is, PASP selectively binds to DNA. The electrostatic interaction or hydrogen bond between PASP and DNA is enhanced, causing the release of more EB dye. However, addition of PASP−cyclen to DNA substantially induced fluorescence decease by up to 54.6% with the same concentration of quenching agent, obtaining C50 = 77.4 μg·mL−1 and Kapp = 2.35 × 107 (Figure 3B). Subsequently, the data are plotted according to the Stern−Volmer equation22 as shown in Figure 3C. DNA-EB is efficiently quenched by PASP−cyclen, resulting in a strict linear plot of a slope of 0.028, which is much larger than that of 0.005 by PASP. A large slope value corresponds to strong quenching and high binding ability to DNA. The result suggests that the interaction between quencher and DNA is enhanced due to the introduction of cyclen, resulting in the higher binding activity than that of PASP skeleton. Conformational changes of DNA interacting with PASP− polyamine conjugates are further verified by CD experiments. All samples to be tested do not show any CD signal in the

studied region studied. Positive and negative ellipticities centered around 245 and 275 nm arise from the DNA itself, revealing a double-stranded structure for the DNA (Figure 4, dashed line). Free DNA shows a typical CD spectrum for Btype. As control, addition of PASP or cyclen with NH2-terminal linker to the DNA solution does not induce any CD signal change, as shown in Figure 4A, which suggests little or no interaction between DNA and ligands. Detectable changes in the CD spectra are, however, observed upon mixing DNA with PASP−cyclen under the same conditions. Both molar ellipticities of DNA at 245 and 275 nm are suppressed, accompanied with a certain quantity of red-shift (Figure 4B, red line). The result could be attributed to the weak interaction between DNA and PASP−cyclen, such as electronic charge and hydrogen bond. The absence of any detectable CD signals at 310−400 nm is the proof of the absence of minor groove binding between DNA and PASP−cyclen. The similar CD signal is observed under the same conditions when PASP−tacn (Figure 4B, purple line) and PASP−cyclam (desert tan line) are used as samples, respectively. These results are consistent with the affinity to DNA in the presence of PASP−polyamine, also shown by fluorescence quenching assay. Control Assays for Exploring the Active Functional Group. In previous reports, no extraordinary activity of DNA double-strand cleavage was found in polyamine or PASP alone, especially to metal-free medium. However, simple polyamine grafted to PASP presents remarkable ability on the two breaks in this work, which could result from the synergy of polyaminegrafted and the remaining carboxyl in PASP. To further elucidate the possible process of DNA cleavage induced by PASP−polyamine, a series of ingenious control compounds 1835

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PSI−cyclen, PASP-cyclen-propyl, and PASP-cyclen-hydroxyethyl are designed and synthesized with the same degree of cyclen substitutes (from the same intermediate PSI−cyclen). The influence of the trace metal in medium and whether polyamine grafted to PASP on cleavage events are considered in detail. The corresponding assays are performed as shown in Figure 5. Initially, 38.2 μg·mL−1 of PASP, cyclen, and Zn(II) as

Figure 6. Possible process of DNA double-strand cleavage by PASP− polyamine.

linker plays an important role in randomicity of linearization. Compared with PASP−polyamine, although lysine−enediyne conjugates presented a good ability of linearization (n1/n2 10 for 32 min), it is worth pointing out that the whole process is achieved with the aid of irradiation. Similar to our cleavage mechanism, the work of Sheng et al.24 indicated DNA cleavage induced by tacn derivatives with guanidinium and hydroxyethyl side arms. The guanidinium group serves to bind and electrophilically activate the anionic phosphodiester through hydrogen bonding and electrostatic interaction, while the hydroxyl group works as a nucleophilic group in the transphosphorylation reaction. Kinetic data of DNA cleavage gave a good kmax of 0.160 h−1, but it showed more randomness double-strand cleavage (n1/n2 from 8.16 to 43.76, calculated from Supporting Information of ref 24) than PASP−polyamine in optimum conditions. The excellent linearization may be attributed to the accumulated active groups on the short peptide skeleton, which provide a more efficient and deliberate attack on both strands.

Figure 5. Quantitation of various DNA forms in control assays. pUC18 plasmid DNA is treated with various compounds (38.2 μg·mL−1) in Tris−HCl (pH 7.4) for 3 h at 37 °C.

independent control react with DNA, respectively, and little or no linearization of plasmid DNA is observed. The combination control of any two agents, such as PASP+cyclen, cyclen+Zn(II), and PASP+Zn(II), present good ability of DNA cleavage, but bad linearization. The remarkable behavior of gel retarding arises when the mixture of three components interacts with DNA, which is attributed to the existence of more electropositive constituents. Further, no detectable DNA cleavage appeared in the presence of PSI−cyclen or PASP-cyclen-propyl without free carboxyl under the same conditions, while PASPcyclen-hydroxyethyl with hydroxyethyl retained the ability of DNA double-strand cleavage in spite of low effectivity, suggesting the necessity of “ungrafted” active moieties. Subsequently, the activity of PASP-Dp with tertiary amine as polyamine moiety is evaluated. From it, no positive result is obtained, which indicates that the multielectropositivity of protonated polyamine plays an important role in cleavage events. Possible Mechanism of DNA Linearization Induced by PASP−Polyamine. Some possible processes of DNA cleavage induced by PASP−polyamine are speculated as shown in Figure 6: (i) the electropositivity of protonated polyamine contributes to the formation of polyamine cation/phosphate anion pair, which is connected via hydrogen bonds and electrostatic interactions. (ii) The electropositive phosphor is liable to be attacked by free ungrafted nucleophilic groups in the PASP skeleton. Phosphodiester linkage of DNA is cleaved by PASP− polyamine via transphosphorylation pathway. (iii) Compared with the free polyamine and nucleophilic group, the grafting mode based on a short peptide skeleton makes it more effective on DNA cleavage due to the accumulation of active groups. Comparison with Analogous Research. A number of cleaving agents have been previously reported to linearize supercoiled DNA in the absence of metal ions. Kovalenko et al.23 reported a type of lysine-enediyne conjugates which suggests a true double-strand DNA cleavage, and the length of

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CONCLUSIONS In conclusion, we synthesized a new family of PASP grafted polyamine conjugates which could act as promising DNA cleavage agents, giving excellent double-strand cleavage ability toward plasmid DNA in the absence of metal ions. The ingenious control assay attempts to prove a possible process in which the free carboxyl groups in the PASP skeleton often serve as a nucleophile, while the polyamine can serve as a protondonating or -accepting group to reduce electron density in phosphate, which is liable to breakage of the phosphodiester bond. Owing to these findings, these short peptides are expected to have exciting applications as mild metal-free catalytic tools in DNA chemistry. Studies are aimed at better understanding of the interaction between short modified peptides and nucleic acids, as well as providing a good strategy for developing novel effective artificial nucleases. The exploration of a definite mechanism and the design of new short peptide-based nucleases with improved DNA site selectivity are underway.

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic route of PASP−polyamine; characteristic via NMR; molecular range via Maldi-Tof MS; comparison of DNA 1836

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(15) Feng, Y. P., Cao, S. L., Xiao, A. S., Xie, W. J., Li, Y. M., and Zhao, Y. F. (2006) Studies on cleavage of DNA by N-phosphoryl branched peptides. Peptides 27, 1554−1560. (16) Sun, M., Ma, Y., Ji, S. H., Liu, H. A., and Zhao, Y. F. (2004) Molecular modeling on DNA cleavage activity of seryl - histidine and related dipeptide. Bioorg. Med. Chem. Lett. 14, 3711−3714. (17) Chen, P. Y., Liu, Y., Gao, X., Xu, N. S, Niu, J., Liu, S. Y., and Zhao, Y. F. (2011) Evaluation of single-stranded oligonucleotide cleavage function of seryl-histidine dipeptide by electrospray ionization mass spectrometry. Phosphorus, Sulfur Silicon Relat. Elem. 186, 933− 935. (18) Nakato, T., Kusuno, A., and Kakuchi, T. (2000) Synthesis of poly(succinimide) by bulk polycondensation of L-aspartic acid with an acid catalyst. J. Polym. Sci., Part A: Polym. Chem. 38, 117−122. (19) Tomida, M., and Nakato, T. (1997) Convenient synthesis of high molecular weight poly(succinimide) by acid-catalysed polycondensation of l-aspartic acid. Polymer 38, 4733−4736. (20) Povirk, L. F., Wubker, W., Kohnlein, W., and Hutchinson, F. (1977) DNA double-strand breaks and alkali-labile bonds produced by bleomycin. Nucleic Acids Res. 4, 3573−3580. (21) Freifelder, D., and Trumbo, B. (1969) Matching of single-strand breaks to form double-strand breaks in DNA. Biopolymers 7, 681−693. (22) Cao, Y., and He, X. W. (1998) Studies of interaction between Safranine T and double helix DNA by spectral methods. Spectrochim. Acta, Part A 54, 883−892. (23) Kovalenko, S. V., and Alabugin, I. V. (2005) Lysine-enediyne conjugates as photochemically triggered DNA double-strand cleavage agents. Chem. Commun. 41, 1444−1446. (24) Sheng, X., Lu, X. M., Zhang, J. J., Chen, Y. T., Lu, G. Y., Shao, Y., Liu, F., and Xu, Q. (2007) Synthesis and DNA cleavage activity of artificial receptor 1,4,7-triazacyclononane containing guanidinoethyl and hydroxyethyl side arms. J. Org. Chem. 72, 1799−1802.

cleavage ability of different derivatives via gel; the content of metal ions in reaction; the TGA data of PASP−polyamine. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+86) 010 64413899, Fax: (+86) 010 82728926, E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support of this research by the National Nature Science Foundation of China is gratefully acknowledged (No. 20972014, No. 21202005, No. 21232005, and No. 21172016.). We thank Dr. Yanxia Jia from Institute of Biophysics, Chinese Academy of Sciences for AFM.

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