Polymer-Induced Perylene Probe Excimer Formation and Selective

Apr 4, 2014 - Wenying Li , Qingfeng Zhang , Huipeng Zhou , Jian Chen , Yongxin Li .... Yongxin Li , Meiding Yang , Huipeng Zhou , Sohail Anjum Shahzad...
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Polymer-Induced Perylene Probe Excimer Formation and Selective Sensing of DNA Methyltransferase Activity through the Monomer− Excimer Transition Yan Wang,†,‡,§ Jian Chen,†,§ Yang Chen,† Wenying Li,†,‡ and Cong Yu*,†,‡ †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ‡ University of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: A new label-free strategy for sensitive fluorometric biosensing based on perylene probe monomer−excimer transition has been developed. A negatively charged perylene probe (compound 1) was used. Compound 1 shows strong monomer fluorescence in an aqueous buffer solution. A cationic polymer could induce aggregation of compound 1 through noncovalent interactions. Compound 1 monomer emission was quenched, and meanwhile strong excimer emission was observed. Upon addition of a single-stranded DNA (an anionic polymer), strong electrostatic attractive interactions between the cationic polymer and the DNA weakened the binding of aggregates of compound 1 to the polycation. Compound 1 monomers were released, and excimer− monomer emission transition was detected. This observation formed the basis for DNA methyltransferase (MTase) activity detection. When the 3′-OH terminus of a duplex DNA was removed, the DNA strands could not be elongated by terminal deoxynucleotidyl transferase (TdT), and little restoration of compound 1 monomer emission was detected. However, in the presence of MTase and endonuclease, the DNA could be specifically methylated and then cleaved into single-stranded fragments. The DNA fragments contained newly generated 3′-OH termini, which could be elongated by TdT. An excimer−monomer transition signal could then be detected. Simple, sensitive, selective, and inexpensive sensing of DNA methylation was therefore established.

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Literature reports have shown that when perylene molecules were brought into close contact with each other, strong excimer emission could be observed.17−19 However, perylene excimer has seldom been used for sensing applications, and covalent labeling with the perylene probe is usually required, which is technically demanding, time-consuming, and expensive.19−21 For instance, when two perylene molecules were covalently incorporated into single-stranded DNA, hybridization of the DNA strands brought the perylene molecules into close contact with each other, and strong excimer emission was observed. The development of a label-free perylene excimer-based biosensing technique is therefore highly desirable. DNA methylation is a biochemical process in which a methyl group is added to cytosine or adenine bases of DNA nucleotides.22,23 It is a crucial epigenetic modification of the genome that is involved in many cellular processes such as DNA replication, mismatch repair, transposition, and gene expression.24,25 A growing number of human diseases have

erylene bisimide (PBI) derivatives have been recognized as excellent dyes and optical materials because of their high fluorescence quantum yield, strong visible light absorption, good chemical inertness, and good photostability and thermal stability.1−5 In addition, the remarkable tendency to form selfassembled aggregates renders them as versatile building blocks for the construction of novel functional supramolecular architectures.6,7 We have observed that a polyanion (such as a single-stranded DNA) could induce strong aggregation of a cationic perylene probe in an aqueous buffer solution. As a result, probe monomer emission was completely quenched. On the basis of this observation, a number of highly sensitive biosensing techniques have been developed.8−10 A number of planar, aromatic compounds such as pyrene can form excimers when an excited-state molecule is brought in close proximity to another ground-state molecule.11−13 Pyrene excimer emission has been utilized in a number of cases for the construction of novel sensing techniques.14−16 Excimer emission offers several distinct advantages. For example, the Stokes shift is usually quite large, which could substantially reduce the background fluorescence at the detection wavelength. The long fluorescence lifetime of excimer emission could also be used for time-resolved biosensing and bioimaging. © 2014 American Chemical Society

Received: January 16, 2014 Accepted: April 4, 2014 Published: April 4, 2014 4371

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been found to be associated with aberrant DNA methylation.26−28 The process of methylation is catalyzed by DNA methyltransferase (MTase) in the presence of S-adenosylmethionine (SAM). Thus, sensing DNA MTase activity and its inhibitor screening are of great importance for fundamental biochemical research, drug discovery, and diagnosis of genetic diseases. Many traditional assay methods, such as gel electrophoresis, high-performance liquid chromatography (HPLC), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), have been developed for the detection of MTase activity.29−33 In recent years, a number of electrochemical, fluorometric, and colorimetric methods for MTase activity detection have been reported.34−42 Fluorometric methods have drawn considerable attention because the assay is convenient and the sensitivity is usually high. However, certain drawbacks still exist. For example, some methods require covalent labeling with a fluorophore or a fluorophore− quencher pair,43−45 which is complicated, time-consuming, and expensive. Some methods are based on the fluorescence turnoff mode, which may produce false-positive output detection signals.36 Therefore, there is an urgent need for development of a label-free, sensitive, selective, and inexpensive fluorometric assay for DNA MTase activity. Terminal deoxynucleotidyl transferase (TdT) is a templateindependent DNA polymerase that can catalyze the repetitive addition of deoxyribonucleotides at the 3′-OH terminus of an oligonucleotide primer in the presence of deoxyribonucleotide triphosphates (dNTPs).46 The primer should be at least three bases long and contain a free 3′-OH terminus.47−52 In this work, we report a new sensing strategy based on polymer-induced perylene probe excimer formation. A negatively charged perylene probe (compound 1, Figure 1)

Scheme 1. Polymer-Induced Formation of Compound 1 Excimer

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EXPERIMENTAL SECTION Instrumentation. Emission spectra were recorded with a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc.). Excitation wavelength was 470 nm and the emission spectra were corrected against photomultiplier tube (PMT) response. The IM/IE value was defined as the intensity ratio of the perylene probe (compound 1) monomer emission (at 548 nm) to excimer emission (at 680 nm). Excitation and emission slit widths were both 7 nm. Quartz cuvettes with 10 mm path length and 2 mm window width were used for the emission measurements. Materials. Compound 1 was prepared as previously described.53 Poly(ethylenimine) (polycation 1, linear, MW 25 000) was purchased from Alfa Aesar (London, UK). Poly(acrylamide-co-diallyldimethylammonium chloride) (polycation 2, 10 wt % in H2O; acrylamide/diallyldimethylammonium chloride = 2.7:1), poly(diallyldimethylammonium chloride) (polycation 3, 35 wt % in H2O, MW < 100 000), and poly(allylamine) (polycation 4, 20 wt % in H2O; MW 17 000) were purchased from Aldrich (St. Louis, MO). Polycation concentrations were defined as the concentration of the repeating unit. SYBR Green I (10000× concentrate) was purchased from Generay Biotech Co. (Shanghai, China). Gentamycin was purchased from Aladdin Reagent Database Inc. (Shanghai, China). All oligonucleotides were synthesized and Ultra-PAGEpurified by Sangon Biological Engineering Technology & Service Co. Ltd. (Shanghai, China) (Table S1, Supporting Information). dam MTase, M.SssI MTase, HpaII MTase, and DpnI were purchased from New England Biolabs, Inc. Terminal deoxynucleotidyl transferase (TdT) was purchased from Sangon Biological Engineering Technology & Service Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and used without further purification. All stock and buffer solutions were prepared with water purified with Milli-Q A10 (Millipore, Billerica, MA). Nucleic acid and enzyme solutions were stored at 4 °C before use. Unless specified, all concentrations of compound 1, polymer, DNA, and buffers are those in the final assay solutions (total sample volume 500 μL). Concentrations of dam MTase, DpnI, SAM, gentamycin, and calf serum were those in the dam MTase enzymatic reaction solution (total sample volume 50 μL). Unless specified, all measurements were performed atambient temperature (22 °C). Duplex DNA (dsDNA-1) was obtained by a simple annealing procedure. Equimolar amounts of the complementary singlestranded nucleic acid strands (DNA-a + DNA-b, final concentration 400 nM each) were mixed in an aqueous buffer solution [2 mM Tris-HAc, 5 mM KAc, and 1 mM Mg(Ac)2, pH 7.9]. The mixture was incubated in a 90 °C water bath for

Figure 1. Structure of perylene probe.

shows strong monomer fluorescence in an aqueous buffer solution. A cationic polymer induced aggregation of compound 1. Compound 1 monomer emission was quenched, and meanwhile significant excimer emission was observed. When a single-stranded DNA (a polyanion) was added, strong electrostatic attractive interactions between the cationic polymer and the DNA weakened the binding of compound 1 aggregate to the polycation. Momomers of compound 1 were released. An excimer−monomer transition was detected (Scheme 1). ON the basis of this observation, a new method for the sensitive detection of DNA MTase activity has been developed. When the 3′-OH termini of a duplex DNA were removed, the DNA strand could not be elongated by TdT, and little restoration of compound 1 monomer emission was detected. However, in the presence of MTase and an endonuclease, the DNA could be specifically methylated and then cleaved into single-stranded fragments. The resulting DNA fragments contained 3′-OH termini, which could be efficiently elongated by TdT. An excimer−monomer transition could therefore be detected. 4372

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concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 μM) were incubated at 37 °C for 2 h. MTase activity was determined as described under Sensing of dam MTase Activity. The relative activity (RA) of dam MTase was defined as

about 10 min and gradually cooled to ambient temperature. The obtained duplex DNA was stored at 4 °C before use. Polycation-Induced Compound 1 Aggregation and Excimer Formation. Different amounts of compound 1 (0.01, 0.1, 1, and 10 μM) were added to a buffer solution of polycation 4 (18 μM) and mixed briefly, and emission spectra were recorded at ambient temperature (total sample volume, 500 μL; buffer, 5 mM Tris-HCl, 5 mM NaCl, and 1 mM MgCl2, pH 7.5). Different amounts of the polycations (0−200 μM polycation 1, 0−40 μM polycation 2, 0−50 μM polycation 3, and 0−28 μM polycation 4) were added individually to buffer solutions of compound 1 (10 μM) and mixed briefly, and emission spectra were recorded at ambient temperature (total sample volume, 500 μL; buffer, 5 mM Tris-HCl, 5 mM NaCl, and 1 mM MgCl2, pH 7.5). Compound 1 Fluorescence Recovery. Different amounts of DNA-15 (0−2.6 μM) were added to a buffer solution of polycation 4 (18 μM) and compound 1 (10 μM). The solutions were mixed briefly and stabilized at 37 °C for 5 min, and then emission spectra were recorded (total sample volume, 500 μL; buffer, 5 mM Tris-HCl, 5 mM NaCl, and 1 mM MgCl2, pH 7.5). Single-stranded DNAs (DNA-15, DNA-25, DNA-30, DNA40, DNA-59, DNA-80, and DNA-100; 200 nM) were individually added to buffer solutions of polycation 4 (18 μM) and compound 1 (10 μM). The solutions were mixed briefly and stabilized at 37 °C for 5 min, and then emission spectra were recorded (total sample volume, 500 μL; buffer, 5 mM Tris-HCl, 5 mM NaCl, and 1 mM MgCl2, pH 7.5). Sensing of dam MTase Activity. A total volume of 50 μL of sample mixture containing 400 nM dsDNA-1, reaction buffer [2.5 μL, 10× buffer: 200 mM Tris-HAc, 500 mM KAc, 100 mM Mg(Ac)2, and 10 mM dithiothreitol (DTT), pH 7.9], 160 μM SAM, 200 units/mL DpnI, and dam MTase of various concentrations (0, 0.2, 0.5, 1, 2, 2.5, 10, 20, 40, and 80 units/ mL) were incubated at 37 °C for 2 h. Sample solutions were incubated at 90 °C for 10 min to inactivate dam MTase and DpnI and then allowed to cool to ambient temperature. Eight units of terminal deoxynucleotidyl transferase (TdT), 0.01 μmol of dNTP, and reaction buffer [5 μL, 10× buffer: 125 mM Tris-HAc, 1 M potassium cacodylate, 5 mM CoCl2, and 0.05% (v/v) Triton X-100, pH 7.2] were added. Sample solutions were incubated at 37 °C for 4 h and then incubated at 90 °C for 10 min to inactivate TdT. Finally, the resulting solutions were added to a solution mixture of compound 1 and polycation 4. The samples were mixed briefly and stabilized at 37 °C for 5 min, and then the emission spectra were recorded (total sample volume, 500 μL; final concentrations, compound 1, 10 μM; polycation 4, 18 μM). dam MTase Activity Assay in Biological Fluid. A total volume of 50 μL of sample mixture containing 5% calf serum, 400 nM dsDNA-1, reaction buffer [2.5 μL, 10× buffer: 200 mM Tris-HAc, 500 mM KAc, 100 mM Mg(Ac)2, and 10 mM DTT, pH 7.9], 160 μM SAM, 200 units/mL DpnI, and dam MTase at various concentrations (0, 20, 40, and 80 units/mL) were incubated at 37 °C for 2 h. dam MTase activity was determined as described under Sensing of dam MTase Activity. dam MTase Inhibitor Screening. A total volume of 50 μL of sample mixture containing 400 nM dsDNA-1, reaction buffer [2.5 μL, 10× buffer: 200 mM Tris-HAc, 500 mM KAc, 100 mM Mg(Ac)2, and 10 mM DTT, pH 7.9], 160 μM SAM, 200 units/ mL DpnI, 20 units/mL dam MTase, and gentamycin at various

RA =

(IM /IE)inhibitor − (IM /IE)0 (IM /IE)no inhibitor − (IM /IE)0

in which (IM/IE)inhibitor and (IM/IE)no inhibitor refer to the IM/IE values of compound 1 in the presence or absence of gentamycin, and (IM/IE)0 refers to background IM/IE value of compound 1.39 Control experiments were carried out to study whether gentamycin had influence on the activity of TdT. First, the DNA methylation assay mixture was prepared by mixing 400 nM dsDNA-1, reaction buffer, 160 μM SAM, 200 units/mL DpnI, and 20 units/mL dam MTase at 37 °C for 2 h, and subsequently the mixture was heated at 90 °C for 10 min (total sample volume, 50 μL). Then 1 μM gentamycin, 0.01 μmol of dNTP, 8 units of TdT, and 5 μL of reaction buffer [10× buffer: 125 mM Tris-HAc, 1 M potassium cacodylate, 5 mM CoCl2, and 0.05% (v/v) Triton X-100, pH 7.2] were added to the reaction mixture, and the sample solution was incubated at 37 °C for 4 h. dam MTase activity was determined as mentioned above.

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RESULTS AND DISCUSSION Cationic Polymer-Induced Perylene Probe Excimer Emission. In an aqueous buffer solution, compound 1 exhibited characteristic monomer emission with peaks at 548 and 587 nm. Different concentrations of compound 1 (0.01, 0.1, 1, and 10 μM) were added to a solution containing 18 μM cationic polymer (polycation 4, Figure 2). Figure 3 shows that

Figure 2. Polycations used in this investigation.

in all cases the perylene probe monomer emission was efficiently quenched. When the concentration of compound 1 was relatively low (0.01 or 0.1 μM), no excimer emission was observed. When the concentration of compound 1 was increased (1 or 10 μM), a new and broad emission band with peak maximum at 680 nm appeared. The new emission band has a very large Stokes shift of about 210 nm. By contrast, compound 1 monomer emission only has a Stokes shift of about 57 nm.53 This new band is attributed to the formation of perylene excimer as a result of polymer-induced aggregation of compound 1 (Figure S1, Supporting Information). The results suggest that excimer emission of compound 1 could be observed only at relatively higher concentrations of compound 1 under our experimental conditions. 4373

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Figure 3. Emission spectra of different concentrations of compound 1 in the absence (black line) or presence (red line) of polycation 4. Assay solutions contained 18 μM polycation 4 and (a) 0.01, (b) 0.1, (c) 1, or (d) 10 μM compound 1.

(IM/IE value) gradually increased upon the gradual increase of DNA-15 concentration. When 1 μM DNA-15 was introduced, the IM/IE value reached its maximum. Further increase of DNA15 concentration caused no further increase in IM/IE value. The results suggest that competitive binding of single-stranded DNA (a polyanion) to the polycation resulted in the release of perylene monomer. In addition, it was observed that the IM/IE value also increased with increasing chain length of singlestranded DNA (Figure 5B and Figure S4 in Supporting Information). The IM/IE value changes could then be directly related to the amount and chain length of the DNA added. This observation formed the basis for the DNA MTase activity detection. MTase Activity Sensing Strategy. DNA adenine methylation (dam) MTase and the restriction endonuclease DpnI were chosen as the model MTase and endonuclease, respectively. The overall sensing strategy is schematically illustrated in Scheme 2. (1) A DNA strand could not be elongated by TdT when the 3′-terminal -OH functional group was removed (3′-ddC). A double-stranded DNA (dsDNA-1, 3′ddC) containing the MTase 5′-G-A-T-C-3′ recognition sequence was used (Table S1, Supporting Information). It could be methylated (5′-G-mA-T-C-3′) by dam MTase in the presence of SAM. DpnI could specifically recognize and cleave the fully methylated G-mA-T-C site. (2) Since dsDNA-1 contained no free 3′-OH functional groups on both strands, it could not be elongated by TdT. In the presence of dam MTase and DpnI, dsDNA-1 was methylated and cleaved. The process produced four single-stranded DNA fragments containing two free 3′-OH termini. The DNA fragments contained the free 3′OH termini could be very much elongated by TdT. (3) When the elongated DNA strands were mixed with polycation− compound 1 complex, competitive binding of DNA to the polycation resulted in the release of perylene monomer. An excimer−monomer transition was detected, and a fluorometric assay for MTase activity was therefore established.

Induced aggregation of compound 1 by four different cationic polymers (polycations 1−4, Figure 2) was studied. The results show that regardless of the cationic polymer used, compound 1 excimer emission could be clearly observed in all cases (Figure 4). The emission intensity ratio (IE/IM value) of the excimer (IE at 680 nm) to the monomer (IM at 548 nm) gradually increased upon gradual increase of polycation concentration. When 160 μM polycation 1, 30 μM polycation 2, 40 μM polycation 3, and 24 μM polycation 4 were introduced, IE/IM values reached their maxima of 10.74, 0.79, 7.67, and 35.96, respectively. Further increase of the polycation concentration caused no further increase in IE/IM value. The results clearly show that the structure of the polymer could significantly influence the strength of excimer emission. While polycation 4 gave the largest IE/IM value of 35.96, polycation 2 could give a maximum IE/IM value of only 0.79. The results suggest that the charge density of the polymer may influence the formation of excimer emission. Polycation 2 has a low charge density, and as a result, its interaction with compound 1 was weaker and its ability to induce aggregation of compound 1 was relatively low.53 The results also show that the concentration of polymer used to reach the maximum IE/IM value was also quite different. Polycation 4 needed only 24 μM; however, polycation 1 needed 160 μM to reach the maximum IE/IM value. Polycation 4 contained protonated amino functional groups, and 1 contained carboxylic acid functional groups. It seems that hydrogen-bonding interactions between the polymer and compound 1 may help to promote excimer formation. Fluorescence Recovery in the Presence of DNA. Figure 5A and Figures S2 and S3 in Supporting Information show that when a single-stranded DNA (DNA-15, 15 bases long; Table S1 in Supporting Information) was added to the solution of polycation 4 (18 μM)−compound 1 (10 μM) complex, significant compound 1 monomer emission recovery was observed. The intensity ratio of monomer to excimer emission 4374

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Figure 5. (A) Changes in IM/IE value of compound 1 upon addition of increasing concentrations of DNA-15 to the sample mixture of compound 1 (10 μM) and polycation 4 (18 μM). (B) Changes in IM/ IE value of compound 1 upon addition of single-stranded DNA (200 nM) of different chain lengths to the sample mixture of compound 1 (10 μM) and polycation 4 (18 μM).

Scheme 2. Schematic Illustration of dam MTase Activity Detection

obvious IM/IE value increase of compound 1 was seen. However, dsDNA-1 with 3′-ddC could not induce similar compound 1 emission changes. The results clearly suggest that DNA without 3′-OH termini could not be elongated by TdT. SYBR Green I is a nucleic acid staining dye. It preferentially binds to duplex DNA rather than single-stranded DNA.54 When SYBR Green I was added to the solution of dsDNA-1, strong emission could be observed. After the enzymatic reaction with dam MTase and DpnI, the emission intensity decreased considerably (Figure S6, Supporting Information). The results indicate dsDNA-1 was initially in the duplex form. In the presence of dam MTase and DpnI, dsDNA-1 was methylated and cleaved and dissociated into four short singlestranded DNA fragments (7−9 bases long).55

Figure 4. Changes in emission spectra (left) and corresponding IE/IM values (right) of compound 1 (10 μM) in the presence of different concentrations of polycations 1−4 (panels a−d, respectively).

Assay Verification and Optimization. Figure S5 (Supporting Information) shows that when 40 nM dsDNA-1OH (15 bases long, with 3′-OH functional group) was added to the solution of polycation 4−compound 1 complex, little IM/IE value increase of compound 1 was observed. In the presence of TdT, dsDNA-1-OH was very much elongated. Thus, an 4375

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The influence of TdT reaction time on the performance of the assay was studied. Figure S7 (Supporting Information) shows that, in the presence of dam MTase, the IM/IE value of compound 1 increased gradually with increasing TdT reaction time. In contrast, in the absence of dam MTase, the IM/IE value remained mostly unchanged within 4 h of the TdT reaction but increased considerably thereafter because of the dNTP background elongation. The TdT reaction time was therefore selected to be 4 h in the assay. Figure 6 shows that, in the presence of 80 units/mL dam MTase, the IM/IE value of compound 1 increased gradually with

Figure 7. (A) Plot of IM/IE value vs dam MTase concentration (0, 0.2, 0.5, 1, 2, 2.5, 10, 20, 40, and 80 units/mL). (Inset) Expanded linear region of the curve. (B) Selectivity of the assay. Columns: (1) blank; (2) M.SssI MTase; (3) HpaII MTase; (4) dam MTase. Enzyme concentrations: 40 units/mL each.

Figure 6. Changes in IM/IE value of compound 1 with dam MTase enzymatic reaction time.

increasing dam MTase enzymatic reaction time. Maximum IM/ IE value was obtained after 2 h of enzymatic reaction. The results indicate that increasing percentage of the substrate strand was gradually methylated by dam MTase and cleaved by DpnI with prolonged enzymatic reaction time. An increasing amount of cleaved strands could be elongated by TdT, so increased IM/IE value was therefore observed. Figure 7A shows that the IM/IE value of compound 1 increased gradually with increasing dam MTase concentration. Our assay method is quite sensitive compared with a previously reported fluorescent method;42−44 the activity of 0.2 unit/mL dam MTase could be easily detected. Selectivity Study. To address the selectivity of our assay, control experiments were conducted. M.SssI MTase and HpaII MTase were selected as the potential interference enzymes. They are also methyltransferases. For example, M.SssI MTase could specifically methylate the cytosine residues within the double-stranded DNA recognition sequence of 5′-C-G-3′.40 Figure 7B shows that 40 units/mL dam MTase could induce a significant IM/IE value increase. In contrast, M.SssI MTase and HpaII MTase at the same concentration could not induce noticeable IM/IE value changes. The results clearly suggest that our assay is highly selective for dam MTase. dam MTase Activity Assay in Biological Fluid. Our assay method was further tested in complex sample mixture containing 5% calf serum. The assay was conducted under the same experimental conditions. Figure S8 (Supporting Information) shows that the more dam MTase is spiked, the larger the IM/IE value of compound 1 obtained. The results suggest that our assay could also be used in complex sample mixtures. Inhibitor Screening. Our assay could also be used for the screening of potential MTase inhibitors. Gentamycin, a wellknown DNA MTase inhibitor,43,56 was tested. The influence of gentamycin on the enzymatic activity of DpnI and TdT was first

investigated. It was reported that gentamycin had no inhibition effect on the activity of DpnI when its concentration was less than 1 μM.56 In addition, control experiments indicate that gentamycin at a concentration of less than 1 μM had hardly any influence on the activity of TdT (Figure S9, Supporting Information). Thus, gentamycin at less than 1 μM concentration was used in our study. Figure 8 shows that the relative

Figure 8. Relative activity of dam MTase vs gentamycin concentration.

activity of dam MTase decreased gradually with increasing gentamycin concentration. The IC50 value was calculated to be 0.512 μM (Figure S10, Supporting Information), which is comparable to the reported literature values.41,56 The results clearly demonstrate that our assay could be used for screening of potential DNA MTase inhibitors. 4376

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CONCLUSIONS In summary, a new sensing strategy based on the perylene probe monomer−excimer transition has been developed. A negatively charged perylene probe (compound 1) showed strong monomer fluorescence in an aqueous buffer solution. A cationic polymer could induce aggregation of the perylene probe through noncovalent interactions. Compound 1 monomer emission was efficiently quenched, and meanwhile strong turn-on excimer emission with a large Stokes shift (about 210 nm) was observed. When single-stranded DNA was added to the mixture of compound 1 and cationic polymer, monomers of compound 1 were released, and an excimer− monomer emission transition was detected. On the basis of this observation, a label-free, sensitive, selective MTase assay has been developed. Our assay has several distinct advantages: first, an excimer emission has large Stokes shift and long fluorescence lifetime, which could considerably reduce the background interference. Second, it is based on the “excimer− monomer transition” mode, which could considerably reduce the possibility of false-positive signals associated with other fluorometric methods. Third, our method is label-free and does not need technically demanding and complicated covalent labeling. We envision that the polymer-induced perylene probe excimer formation could be employed for the development of novel sensing techniques for the detection of various biomolecules and could be used for biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

Ten figures showing UV/vis absorption spectra of compound 1 in the presence and absence of polycation 4, changes in emission spectrum of compound 1 upon addition of DNA-15, changes in IM/IE value of compound 1 in the absence and presence of DNA-15, changes in emission spectrum of compound 1 upon addition of single-stranded DNA, DNA elongation by TdT, emission spectra of SYBR Green I, changes in IM/IE value of compound 1 with TdT reaction time in the presence and absence of dam MTase and with dam MTase concentration in 5% calf serum, influence of 1 μM gentamycin on TdT activity, and inhibition efficiency of dam MTase versus gentamycin concentration; one table listing oligonucleotides used. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; fax +86-431-85262710. Author Contributions §

Y.W. and J.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the “100 Talents” program of the Chinese Academy of Sciences, the National Basic Research Program of China (973 Program, 2011CB911002), the National Natural Science Foundation of China (21275139, 91027036, 21075119), the Pillar Program of Changchun Municipal Bureau of Science and Technology (2011225), and the Open Project of the State Key Laboratory of Supramolecular Structure and Materials (Grant SKLSSM201415). 4377

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