Estimation of Binding Constants of Peptide Nucleic Acid and

Publication Date (Web): May 24, 2012 .... average of the maximum values in the first derivative of the melting curves obtained from the heating and co...
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Technical Note pubs.acs.org/ac

Estimation of Binding Constants of Peptide Nucleic Acid and Secondary-Structured DNA by Affinity Capillary Electrophoresis Lal Mohan Kundu,†,‡ Harumi Tsukada,†,§ Yukiharu Matsuoka,†,§ Naoki Kanayama,† Tohru Takarada,*,† and Mizuo Maeda†,§ †

Bioengineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Department of Advanced Materials Science, School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwano-ha, Kashiwa, Chiba 277-8561, Japan

§

S Supporting Information *

ABSTRACT: An affinity capillary electrophoresis method was developed to determine a binding constant between a peptide nucleic acid (PNA) and a hairpin-structured DNA. A diblock copolymer composed of PNA and polyethylene glycol (PEG) was synthesized as a novel affinity probe. The base sequence of the probe’s PNA segment was complementary to a hairpinstructured region of a 60-base single-stranded DNA (ssDNA). Upon applying a voltage, the DNA hairpin migrated slowly compared to a random sequence ssDNA in the presence of the PNA probe. This retardation was induced by strand invasion of the PNA into the DNA hairpin to form a hybridized complex, where the PEG segment received a large amount of hydrodynamic friction during electrophoresis. The binding constant between the PNA probe and the DNA hairpin was easily determined by mobility analysis. This simple method would be potentially beneficial in studying binding behaviors of various artificial nucleotides to natural DNA or RNA.

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invade the interior regions of mixed-sequence, double helical BDNA. To exploit these unique features of PNA to detect a specific sequence within a folded DNA analyte through a rational design of the PNA sequence, thermodynamic data should be collected about hybridization of PNA and various secondary-structured DNAs. A general platform that allows rapid and facile determination of these data is strongly required because a conventional method based on melting curve measurements is time-consuming and labor-intensive.14 This paper describes an affinity capillary electrophoresis (ACE) method using a polymer-modified PNA as a novel affinity probe to perform a thermodynamic study of the interaction between PNA and secondary-structured DNA. We have thus far developed an ACE method for single-nucleotide polymorphism genotyping by use of a diblock copolymer probe composed of an allele-specific oligonucleotide and a synthetic polymer with water solubility and charge neutrality: polyethylene glycol (PEG)31−34 or polyacrylamide.35 During electrophoresis of a mixture of the target and nontarget ssDNA, the affinity probe binds selectively to the target ssDNA and thus decreases the electrophoretic mobility by exerting a large amount of hydrodynamic friction. Importantly, the binding constant between the target ssDNA and the probe was easily determined by mobility analysis.35 In the present study, we synthesize a novel block copolymer probe composed

arge subsets of analytical methods for detecting a specific nucleic acid sequence, such as real-time PCR and DNA microarrays, involve a hybridization step between a singlestranded DNA (ssDNA) analyte and an allele-specific oligonucleotide probe.1 However, ssDNA analytes often exhibit secondary structures under usual analytical conditions. When a sequence of interest is located in a folded region of the analyte, the duplex formation between the ssDNA analyte and the oligonucleotide probe is kinetically unfavorable.2−4 For example, Fertig and co-workers reported that the detection sensitivity on a DNA microarray decreased with increasing thermal stability of the analyte’s hairpin structure.5 To accelerate the probe’s hybridization to the secondary-structured analyte, Kolpashchikov and co-workers have recently developed binary molecular beacon probes.6,7 Alternatively, an oligonucleotide analogue such as peptide nucleic acid (PNA) can serve as a strong affinity probe for the folded DNA analytes. PNA consists of the natural nucleobases tethered to an electrically neutral polyamide backbone.8 By virtue of the absence of electrostatic repulsion between the two strands, the thermal stability of the PNA/DNA heteroduplex is significantly higher than that of the parent DNA/DNA homoduplex. For this reason, PNA strongly hybridizes to the secondary-structured DNA through strand invasion.9−12 Earlier studies showed that PNA invaded DNA hairpins,13,14 duplex termini,15 homopurine-16 and homopyrimidine-regions within dsDNA,17 supercoiled plasmids,18 and chromosomal DNAs.19 In addition, base- and/or backbone-modified PNA20−28 or unmodified PNA with an ssDNA-binding protein29,30 can also © 2012 American Chemical Society

Received: April 18, 2012 Accepted: May 24, 2012 Published: May 24, 2012 5204

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Technical Note

of an allele-specific PNA and PEG (PEG-b-PNA) to estimate a thermodynamic parameter for hybridization of a conventional PNA and a hairpin-structured ssDNA in a similar manner. Various research groups have successfully utilized PNA as an affinity ligand in ACE systems.36−41 To the best of our knowledge, however, PNA invasion into folded ssDNA analytes has never been used in ACE techniques. The present method would be potentially beneficial in studying binding behaviors of various artificial nucleotides to natural DNA or RNA.



MATERIALS AND METHODS Reagents. All reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) unless otherwise noted. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Pierce (Rockford, IL). All PNAs and 5′FITC-labeled ssDNAs were obtained from Fasmac (Kanagawa, Japan) and Tsukuba Oligo Service (Ibaraki, Japan), respectively, and were used without further purification. The PNA and DNA concentrations were determined by measuring the absorbance at 260 nm with a UV 2550 spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan). Maleimideterminated methoxy PEG (mPEG-MAL; Mn = 5500, Mw/Mn = 1.03) was purchased from Nektar Therapeutics (San Carlos, CA). Deionized water (>18.1 MΩ) purified with a Milli-Q instrument from Millipore (Billerica, MA) was used for all experiments. DNA Samples. Chemically synthesized 5′-FITC-labeled 60base DNA strands (HP2w and HP3w) and their single-basesubstituted mutants (HP2m and HP3m) were used as model analytes. The base sequences are shown in Figure 1a and Figure S1 (Supporting Information). HP2w and HP2m correspond to a fragment of the mitochondrial cytochrome b gene of cucumber powdery mildew and its single-base mutant at codon 143 (GGT to GCT), respectively. Similarly, HP3w and HP3m correspond to a part of the same gene of wheat powdery mildew and its mutant, respectively. This single-base substitution induces strobilurin-related fungicide resistance.42 As an internal standard (IS), a 5′-FITC-labeled 60-base ssDNA with a random sequence was added to the sample. In addition, 5′-FITC-labeled 60-base ssDNAs exhibiting a defined hairpin structure with a 3-base loop and a 2- to 7-base pair stem (HP2−HP7) were also used as model analytes. The folded structure as well as its thermal and thermodynamic parameters of the DNA hairpin was calculated using mfold.43 Affinity Probes. PEG-b-PNA was synthesized via the Michael addition reaction of the cysteine-terminated PNA to the mPEG-MAL in the presence of TCEP as a reductant. Typical procedures were as follows. PNA (200 μM), mPEGMAL (10 mM), and TCEP (10 mM) were dissolved in 10 mM Tris-HCl buffer (pH 7.4). The mixture was deoxidized by bubbling dry nitrogen gas for 10 min and was allowed to react overnight at room temperature. The product was purified using a gel-filtration column packed with Sephadex G-100 gel (GE Healthcare, Buckinghamshire, UK) to remove unreacted PNA and TCEP. Further purification was carried out using a cationexchange Vivapure S Mini H spin column from Sartorius Stedim Biotech (Aubagne, France) to remove excess mPEGMAL. The elution profile was monitored by measuring the absorbance at 260 nm with a microplate reader SpectraMax Plus 384 from Molecular Devices (Sunnyvale, CA). Appropriate fractions were collected and dialyzed against deionized water (Mw cutoff, 500). The final solution was lyophilized to obtain PEG-b-PNA. The yield was determined to be 20% by

Figure 1. (a) Sequences of the ssDNA analytes (in part) and the affinity probes used in this study. All of the sequences are FITClabeled at the 5′-terminus. The probe-binding site of the analytes is shown in red. The single-base substitution site is underlined. Total sequences of the analytes are given in Figure S1 (Supporting Information). (b) Chemical structure of PEG-b-PNA used in this study. (c) The secondary structure around the probe-targeting region of HP2w and HP3w predicted using mfold. A dashed line indicates base pairing.

measuring the absorbance at 260 nm. For comparison, a block copolymer composed of the same mPEG-MAL and the DNA counterpart (PEG-b-DNA) was prepared by the reported method.31 Capillary Electrophoresis. Capillary electrophoresis (CE) was performed on a P/ACE MDQ system with a laser-induced fluorescent (LIF) detector from Beckman-Coulter (Fullerton, CA). The capillary tube used was a polyacrylamide-coated capillary (internal diameter, 50 μm; total length, 50.5 cm; effective length, 40.5 cm) from Beckman-Coulter. This tube was used to suppress an electroosmotic flow. As a running buffer, 50 mM Tris-borate buffer (pH 7.4) containing 10 mM NaCl and 0.50 mM MgCl2 was used. The fluorescence of FITC-labeled sample DNA was measured with excitation at 488 nm and recording emission at 520 nm. The temperature of the capillary tube was held constant (25 °C) with a recirculating liquid coolant system. Prior to electrophoresis, a solution of the affinity PNA probe (100−500 nM) in the running buffer was injected into the capillary tube from the cathode end by positive pressure (20 psi for 45 s). Next, the running buffer solution of the analyte(s) and IS (0−20 nM each) was introduced into the capillary tube using a similar method (0.50 psi for 10 s). Electrophoresis was performed under reversed polarity with a constant voltage of −15 kV for 20 min. Between measurements, the capillary tube was sufficiently washed with the running buffer (20 psi for 30 min) and then filled with the running buffer containing the probe (20 psi for 1.0 min). Each CE was carried out in triplicate to confirm the reproducibility. The mobility analysis was conducted by the reported method 5205

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using a Lineweaver−Burk-type plot (Supporting Information).35 Melting Temperature. Melting curves of the folded ssDNA (1.0 μM) in 50 mM Tris-borate buffer (pH 7.4) containing 10 mM NaCl and 0.50 mM MgCl2 were obtained by measuring the change of absorbance at 260 nm as a function of temperature. A UV-2550 spectrophotometer equipped with a TMSPC-8 temperature controller unit (Shimadzu) was used. The heating and cooling ramp was 1.0 °C/min. The sampleholding chamber was kept flushed with dry N2 gas during the measurements. The melting temperature (Tm) was determined as an average of the maximum values in the first derivative of the melting curves obtained from the heating and cooling processes. Each melting experiment was performed in triplicate to confirm the reproducibility.



RESULTS AND DISCUSSION Synthesis of the Affinity Probe. PEG-b-PNA was synthesized via the Michael addition reaction of the cysteineterminated PNA to the maleimide-modified PEG. The chemical structure of PEG-b-PNA is illustrated in Figure 1b. The sequence of the PNA segment was complementary to a part of HP2w and HP3w, including a single-base substitution site (Figure 1a). To enhance the water solubility of the PNA segment, four consecutive lysine residues were inserted between the N-terminal cysteine and the 7-base PNA.44 The product was characterized by measuring the molecular weight with size exclusion chromatography (Supporting Information). The weight-averaged molecular weight (Mw), the numberaveraged molecular weight (Mn), and the polydispersity index (Mw/Mn) of the copolymer are given in Table S1 (Supporting Information). The observed molecular weight agreed fairly well with the calculated one. Capillary Electrophoresis of the Folded DNA with the PEG-b-DNA Probe. We initially examined the ability of the previous DNA-based probe to hybridize to the folded ssDNA analytes. Figure 2a shows the electropherogram of a mixture of HP2w and HP2m when PEG-b-DNA was used as a probe. Two peaks were observed; the peak at 8.7 min was assigned to HP2m and that at 10.0 min was assigned to HP2w. The retardation of the HP2w peak compared to the HP2m peak was due to the formation of the affinity complex between HP2w and the probe via hybridization during electrophoresis.33 In contrast, the HP3w and HP3m peaks were unresolved around 8.8 min under the same conditions (Figure 2b). This means that the hybridization between HP3w and PEG-b-DNA was strongly inhibited. The disappearance of the affinity is attributable to self-folding of HP3w at the target region. This is induced by its flanking region, which is different from that of HP2w. To test this hypothesis, we first measured the melting curves of HP2w and HP3w (Figure S2, Supporting Information). The Tm values of HP2w and HP3w were determined to be 15.3 and 26.3 °C, respectively. As predicted, HP3w had a folded structure(s) showing higher thermal stability than that of HP2w. We next calculated the secondary structures by mfold;43 the results given in Figure S3 (Supporting Information) suggested that probe-targeting regions of both HP2w and HP3w were involved in the hairpin structures. Figure 1c shows the most stable secondary structure predicted for the probe-binding site of HP2w with Tm of 26.3 °C and that of HP3w with Tm of 43.6 °C. It can be seen that the probe-targeting region of HP3w bears a hairpin structure with higher stability than that of HP2w. It is worth pointing out

Figure 2. Electropherograms showing the separation of (a) HP2w and HP2m with PEG-b-DNA, (b) HP3w and HP3m with PEG-b-DNA, and (c) HP3w, HP3m, and IS with PEG-b-PNA. Conditions: running buffer, 50 mM Tris-borate (pH 7.4) containing 10 mM NaCl and 0.50 mM MgCl2; capillary temperature, 25 °C; applied voltage, −15 kV; (a, b) [probe] = 5.0 μM; [analyte] = 50 nM each; (c) [probe] = 500 nM; [analyte] = 20 nM each; [IS] = 20 nM.

that the differences in the hairpin structure and its thermal stability are induced by a single-nucleotide substitution in the vicinity of the probe-binding site (HP2w: AACT; HP3w: AACC). Taken together, these results suggest that the hybridization between HP3w and PEG-b-DNA during electrophoresis was unfavorable due to the folded structure exhibited by the probe-binding region of HP3w. Capillary Electrophoresis of the Folded DNA with the PEG-b-PNA Probe. We next used a new PEG-b-PNA probe. Figure 2c shows the electropherogram of a mixture of HP3w and HP3m with PEG-b-PNA. As expected, the migration time of HP3w was found to be much longer than that of HP3m. Moreover, the migration time of HP3m was also longer than that of IS. These results indicate that a strong affinity occurred between PEG-b-PNA and HP3w, but a weak affinity also appeared between the probe and HP3m. In contrast, the retardation of the HP3w and HP3m peaks was not observed when the PNA segment only or the PEG segment only was used (Figure S4, Supporting Information). Therefore, a linkage between two segments was required to achieve a good separation. Figure 3 shows a schematic diagram illustrating the electrophoresis of the DNA hairpin and IS in the presence of the PEG-b-PNA probe. Both ssDNAs migrate toward the anode end in response to voltage applied across the capillary 5206

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Figure 3. Schematic diagram illustrating the affinity capillary electrophoresis of the DNA hairpin and the internal standard DNA (IS) in the presence of the PEG-b-PNA probe. For clarity, the slow migration of the probe from anode to cathode is not shown.

hybridized complex with PEG-b-PNA for almost all of the total migration time. For comparison, we also determined the RC/RD and K values for the complex of PEG-b-DNA and HP2w in a similar manner. Interestingly, the RC/RD value for PEG-b-DNA and HP2w was almost identical to that for PEG-b-PNA and HP3w. This is because the amount of hydrodynamic friction that the PEG segment received within the complex between PEG-b-PNA and HP3w was almost the same as that between PEG-b-DNA and HP2w. On the other hand, the K value for the complex between PEG-b-PNA and HP3w was about 280 times larger than that between PEG-b-DNA and HP2w. Similarly, we determined the fraction of migration time for the complex between PEG-b-DNA and HP2w to be 60%, as shown in Figure 2a. Hybridization of PNA and the DNA Hairpin. The strong affinity between PEG-b-PNA and HP3w suggests the hybridization of the probe’s PNA segment and the folded target region of HP3w. As shown in Figure S3 (Supporting Information), however, the probe-binding region of HP3w could produce at least one structural isomer of the hairpin structure shown in Figure 1c, and both structures may coexist at equilibrium during the electrophoresis. Because the experimental determination of the HP3w’s solution structure was beyond the scope of this paper, we instead used different ssDNA analytes with a well-defined hairpin structure (HP2− HP7) in order to answer the question of whether the PEG-bPNA probe could invade the stably folded ssDNA. Figure 4a shows the analyte’s sequences as well as the secondary structures together with the Tm values calculated by mfold.43 The Tm values were also experimentally determined from the melting curves of truncated 21-base ssDNAs (Figure S6, Supporting Information). There was a fairly close agreement between the calculated and observed Tm values for HP3−HP7 (Table S2, Supporting Information), indicating the accuracy of the prediction that HP3−HP7 exhibited a hairpin structure consisting of a 3-base loop and a 3- to 7-base pair stem. Figure 4b shows the electropherograms of the DNA hairpins with the previous PEG-b-DNA probe. By increasing the stem’s base pair number from 2 to 4 (HP2−HP4), the retardation of the analyte peak from the IS peak was gradually diminished. In particular, the HP4−HP7 and IS peaks overlapped completely, suggesting that the folding of the analyte greatly inhibited the complex formation with PEG-b-DNA. Figure 4c shows the electropherograms of the same DNA hairpins with PEG-bPNA. Interestingly, all the hairpin’s peaks were considerably retarded from the IS peak, which strongly suggests that PEG-b-

tube. The DNA hairpin migrates slowly because it forms a fully matched duplex with the probe in equilibrium during electrophoresis. The PNA segment can invade the secondarystructured region of the DNA analyte to form a hybridized complex exhibiting drastically reduced electrophoretic mobility (μC) compared to that of free ssDNA (μD). This is because the long, electrically neutral PEG segment receives a large amount of hydrodynamic friction.45,46 The effect of PEG is analogous to that of a parachute attached to a moving object. HP3m migrates faster than HP3w because the probe’s binding is weak due to the single-base mismatch, resulting in a smaller fraction of migration time for the complex of HP3m than for that of HP3w. IS migrates most rapidly without binding to the probe; the mobility is considered to be μD. As a result, the mixture of HP3w, HP3m, and IS is separated, and each fluorescently labeled analyte is detected at the anode end by a LIF detector in the following order: IS, HP3m, and HP3w. Mobility Analysis. Using a Lineweaver−Burk-type analysis, we determined both the mobility (μC) and the binding constant (K) of the complex formed between HP3w and PEG-b-PNA during electrophoresis (Figure S5, Supporting Information). The ratio of the hydrodynamic radius of the complex to that of the free ssDNA (RC/RD), which was calculated using eq S4 (Supporting Information), was also determined. The results are listed in Table 1. Substituting the K value of the complex between PEG-b-PNA and HP3w into eq S2 (Supporting Information), we determined the fraction of migration time for the complex between PEG-b-PNA and HP3w to be 98%, as shown in Figure 2c. This means that HP3w migrated as the Table 1. Electrophoretic Mobility of the Complex (μC) between the Probe and the Analyte, Ratio of the Hydrodynamic Radius of the Complex (RC) to That of the Free ssDNA (RD), and Binding Constant (K) of the Complexa probe/analyte PEG-b-PNA/ HP3w PEG-b-DNA/ HP2w PEG-b-PNA/ HP5

μC (10−4 cm2/(Vs))

μD (10−4 cm2/(Vs))

RC/RDb

−1.79

−2.81

1.46

−2.04

−2.65

1.45

0.298

−1.73

−2.63

1.42

7.82

K (106 M−1) 83.8

a Determined in 50 mM Tris-borate buffer (pH 7.4) containing 10 mM NaCl and 0.50 mM MgCl2. bCalculated using eq (S4) (Supporting Information) with μC and μD.

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Figure 4. (a) Sequences of the hairpin-structured ssDNA analytes. Shown are the secondary structure and its melting temperature in parentheses, predicted using mfold. Note that the terminal base pair (T-A) adjacent to the loop might be thermodynamically unstable (breathing), thus causing a temporal increase of the loop’s base-number from 3 to 5. (b, c) Electropherograms showing the mobility of the hairpin-structured analytes (b) with PEG-b-DNA and (c) with PEG-b-PNA. Conditions: (b) [probe] = 5.0 μM; [HP] = 50 nM; [IS] = 50 nM; (c) [probe] = 500 nM; [HP] = 5.0 nM; [IS] = 5.0 nM. The other conditions are the same as in Figure 2.

reported value for the 8-base pair PNA/DNA duplex between the conventional PNA and the DNA hairpin with a 4-base loop and a 4-base pair stem (ΔG(298K) = −8.0 kcal/mol).14 Substituting the K value obtained here into eq S2 (Supporting Information), we determined that the fraction of migration time for the complex of HP5 and PEG-b-PNA was 80%. This means that HP5 migrated as a complex for 80% of the total migration time and as the free ssDNA for 20%. It is thus clearly indicated that the reversible hybridization of the PEG-b-PNA probe and the hairpin-structured DNA analyte took place during electrophoresis.

PNA disrupted the folded structure and formed the affinity complex (Supporting Information). While the retardations of the HP0−HP5 peaks were quite similar to each other, the retardation of the HP5−HP7 peaks was gradually suppressed by increasing the stem’s base pair number from 5 to 7. Presumably the end-capping of the probe-targeting region within the stem by the G−C base pair(s) at the side opposite the loop decreased the invasion efficiency for HP6 and HP7. This observation is consistent with the fact that a conventional PNA invades dsDNA termini but hardly disrupts the interior region of mixed-sequence dsDNA.15 Using the same Lineweaver−Burk-type analysis, we determined the RC/RD and K values of the complex between PEG-b-PNA and HP5 (Table 1 and Figure S5, Supporting Information). To our knowledge, the present method has enabled the first investigations concerning the thermodynamics of hybridization between PNA and the DNA hairpin through an approach other than the conventional melting curve measurements.14 The validity of the mobility analysis was supported by the following three comparisons. First, the RC/RD value of the complex between PEG-b-PNA and HP5 was almost the same as that between PEG-b-PNA and HP3w. This agrees well with the formation of the commonly sized complex consisting of the 60-base ssDNA analyte and the same PEG-bPNA probe. Second, the K value of the complex between PEGb-PNA and HP5 was 1 order of magnitude smaller than that of the complex between PEG-b-PNA and HP3w. This is in line with the observation that the Tm value for the folding of HP5 (70.3 °C, Table S2, Supporting Information) was remarkably higher than that of HP3w (26.3 °C). Finally, the Gibbs free energy change calculated using the observed K value for the 7base pair PNA/DNA duplex between the PEG-b-PNA and the HP5 composed of a 3-base loop and a 5-base pair stem (ΔG(298K) = −9.4 kcal/mol) was roughly comparable to the



CONCLUSION



ASSOCIATED CONTENT

This study demonstrated that the PEG-b-PNA probe invaded a secondary-structured ssDNA analyte to form a hybridized complex during electrophoresis, allowing for evaluation of a thermodynamic parameter (ΔG) for hybridization of PNA to the DNA hairpin. The present method is of greater advantage than the preexisting one based on melting curve measurements because the present method needs a smaller amount of PNA (2 pmol for each run). It should be further emphasized that this analysis requires a measurement time as short as 20 min. Hence, this ACE could potentially serve as a general platform for studying the dynamic binding behaviors of various artificial nucleotides to natural DNA or RNA.

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 5208

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(28) Bahal, R.; Sahu, B.; Rapireddy, S.; Lee, C. M.; Ly, D. H. ChemBioChem 2012, 13, 56−60. (29) Ishizuka, T.; Otani, K.; Sumaoka, J.; Komiyama, M. Chem. Commun. 2009, 45, 1225−1227. (30) Ishizuka, T.; Tedeschi, T.; Corradini, R.; Komiyama, M.; Sforza, S.; Marchelli, R. ChemBioChem 2009, 10, 2607−2612. (31) Kanayama, N.; Takarada, T.; Kimura, A.; Shibata, H.; Maeda, M. React. Funct. Polym. 2007, 67, 1373−1380. (32) Kanayama, N.; Takarada, T.; Kimura, A.; Shibata, H.; Maeda, M. J. Sep. Sci. 2008, 31, 837−844. (33) Kanayama, N.; Takarada, T.; Shibata, H.; Kimura, A.; Maeda, M. Anal. Chim. Acta 2008, 619, 101−109. (34) Tsukada, H.; Watanabe, T.; Kanayama, N.; Takarada, T.; Maeda, M. Electrophoresis, in press. (35) Kanayama, N.; Shibata, H.; Kimura, A.; Miyamoto, D.; Takarada, T.; Maeda, M. Biomacromolecules 2009, 10, 805−813. (36) Perry-O’Keefe, H.; Yao, X. W.; Coull, J. M.; Fuchs, M.; Egholm, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14670−14675. (37) Igloi, G. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8562−8567. (38) Basile, A.; Giuliani, A.; Pirri, G.; Chiari, M. Electrophoresis 2002, 23, 926−929. (39) Tedeschi, T.; Chiari, M.; Galaverna, G.; Sforza, S.; Cretich, M.; Corradini, R.; Marchelli, R. Electrophoresis 2005, 26, 4310−4316. (40) Grosser, S. T.; Savard, J. M.; Schneider, J. W. Anal. Chem. 2007, 79, 9513−9519. (41) Savard, J. M.; Grosser, S. T.; Schneider, J. W. Electrophoresis 2008, 29, 2779−2789. (42) Ishii, H.; Fraajie, B. A.; Sugiyama, T.; Noguchi, K.; Nishimura, K.; Takeda, T.; Amano, T.; Hollomon, D. W. Phytopathology 2001, 91, 1166−1171. (43) Zuker, M. Nucleic Acids Res. 2003, 31, 3406−3415. (44) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895−1897. (45) Meagher, R. J.; Won, J. I.; McCormick, L. C.; Nedelcu, S.; Bertrand, M. M.; Bertram, J. L.; Drouin, G.; Barron, A. E.; Slater, G. W. Electrophoresis 2005, 26, 331−350. (46) Grass, K.; Holm, C.; Slater, G. W. Macromolecules 2009, 42, 5352−5359.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ‡

Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Strategic Programs for R&D at RIKEN (to T. T.) and by a Grant-in-Aid for Scientific Research (A) (No. 20245020) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M. M. and T. T.).



REFERENCES

(1) Syvänen, A. C. Nat. Rev. Genet. 2001, 2, 930−942. (2) Sekar, M. M. A.; Bloch, W; St. John, P. M. Nucleic Acids Res. 2005, 33, 366−375. (3) Gao, Y.; Wolf, L. K.; Georgiadis, R. M. Nucleic Acids Res. 2006, 34, 3370−3377. (4) Chen, C.; Wang, W.; Wang, Z.; Wei, F.; Zhao, X. S. Nucleic Acids Res. 2007, 35, 2875−2884. (5) Tan, J. C.; Patel, J. J.; Tan, A.; Blain, J. C.; Albert, T. J.; Lobo, N. F.; Ferdig, M. T. Genomics 2009, 93, 543−550. (6) Grimes, J.; Gerasimova, Y. V.; Kolpashchikov, D. M. Angew. Chem., Int. Ed. 2010, 49, 8950−8953. (7) Nguyen, C.; Grimes, J.; Gerasimova, Y. V.; Kolpashchikov, D. M. Chem.Eur. J. 2011, 17, 13052−13058. (8) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624−630. (9) Nielsen, P. E. Curr. Opin. Mol. Ther. 2010, 12, 184−191. (10) Nielsen, P. E. ChemBioChem 2010, 11, 2073−2076. (11) Armitage, B. A. Drug Discovery Today 2003, 8, 222−228. (12) Demidov, V. V.; Frank-Kamenetskii, M. D. Trends Biochem. Sci. 2004, 29, 62−71. (13) Ørum, H.; Nielsen, P. E.; Jorgensen, M.; Larsson, C.; Stanley, C.; Koch, T. Biotechniques 1995, 19, 472−480. (14) Kushon, S. A.; Jordan, J. P.; Seifert, J. L.; Nielsen, H.; Nielsen, P. E.; Armitage, B. A. J. Am. Chem. Soc. 2001, 123, 10805−10813. (15) Smolina, I. V.; Demidov, V. V.; Soldatenkov, V. A.; Chasovskikh, S. G.; Frank-Kamenetskii, M. D. Nucleic Acids Res. 2005, 33, e146. (16) Egholm, M.; Nielsen, P. E.; Buchardt, O.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 9677−9678. (17) Nielsen, P. E.; Christensen, L. J. Am. Chem. Soc. 1996, 118, 2287−2288. (18) Bentin, T.; Nielsen, P. E. Biochemistry 1996, 35, 8863−8869. (19) Janowski, B. A.; Kaihatsu, K.; Huffman, K. E.; Schwartz, J. C.; Ram, R.; Hardy, D.; Mendelson, C. R.; Corey, D. R. Nat. Chem. Biol. 2005, 1, 210−215. (20) Kaihatsu, K.; Braasch, D. A.; Cansizoglu, A.; Corey, D. R. Biochemistry 2002, 41, 1118−11125. (21) Ishizuka, T.; Yoshida, J.; Yamamoto, Y.; Sumaoka, J.; Tedeschi, T.; Corradini, R.; Sforza, S.; Komiyama, M. Nucleic Acids Res. 2008, 36, 1464−1471. (22) Miyajima, Y.; Ishizuka, T.; Yamamoto, Y.; Sumaoka, J; Komiyama, M. J. Am. Chem. Soc. 2009, 131, 2657−2662. (23) Corradini, R.; Sforza, S.; Tedeschi, T.; Totsingan, F.; Manicardi, A.; Marchelli, R. Curr. Top. Med. Chem. 2011, 11, 1535−1554. (24) Rapireddy, S.; He, G.; Roy, S.; Armitage, B. A.; Ly, D. H. J. Am. Chem. Soc. 2007, 129, 15596−15600. (25) Chenna, V.; Rapireddy, S.; Sahu, B.; Ausin, C.; Pedroso, E.; Ly, D. H. ChemBioChem 2008, 9, 2388−2391. (26) He, G.; Rapireddy, S.; Bahal, R.; Sahu, B.; Ly, D. H. J. Am. Chem. Soc. 2009, 131, 12088−12090. (27) Rapireddy, S.; Bahal, R.; Ly, D. H. Biochemistry 2011, 50, 3913− 3918. 5209

dx.doi.org/10.1021/ac301025m | Anal. Chem. 2012, 84, 5204−5209