Thermodynamics-based Rational Design of DNA Block Copolymers

The number-average molecular weight (Mn) and the molecular weight distribution (Mw/Mn) of the probes for WT1/MT1 were determined by aqueous size ...
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Thermodynamics-based Rational Design of DNA Block Copolymers for Quantitative Detection of Single-Nucleotide Polymorphisms by Affinity Capillary Electrophoresis Ayumi Kimura,†,‡ Naoki Kanayama,† Atsushi Ogawa,†,§ Hideaki Shibata,† Hideo Nakashita,† Tohru Takarada,*,† and Mizuo Maeda†,‡ †

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

‡

S Supporting Information *

ABSTRACT: Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-basemutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe’s ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 106 M−1. The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation.

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perfectly suited to their quantification. Therefore, a more facile and precise method is still required to estimate SNP allele frequency. In general, electrophoretic separation is quite effective for identifying polyelectrolyte nucleic acids. For SNP analysis, diblock copolymers composed of an allele-specific ODN segment and a synthetic polymer segment can work well as an affinity probe in free-solution capillary electrophoresis (CE).5 Water-soluble and electrically neutral polymers, such as poly(ethylene glycol) (PEG)6,7 and polyacrylamide,8 are applicable to the synthetic polymer segment. The separation diagram is shown in Scheme 1a. When the allele-specific ODN segment is designed to be complementary to part of a wild-type ssDNA analyte (WT) including the SNP site, the WT migrates more slowly than its single-base-substituted mutant (MT). This is because the WT encounters a large amount of hydrodynamic friction by forming a reversible complex with the probe. In contrast, a negligibly weak interaction takes place between the MT and the probe owing to a single-base mismatch. The mobility of the MT is almost identical to that of an internal standard ssDNA (IS) with a random sequence. Consequently, a single, overlapping peak for the IS and MT appears earlier than

ingle-nucleotide polymorphisms (SNPs) are genetic variations resulting from single-base substitution in the genomes of living organisms. Distinct categories of SNPs can account for drastic differences in phenotypes. For example, a number of SNPs associated with fungicide resistance have been identified in plant pathogenic fungi.1 The occurrence rate for fungicide-resistant isolates can be precisely evaluated by estimating the SNP allele frequency. This value is determined using a DNA pool,2 which is constructed by mixing equal amounts of genomic DNA extracted from the fungi in a cultivated field. Reliable validation of the occurrence rate could help to avoid the application of ineffective fungicides, which would be of both economic and ecological benefit. As such, not only detection but also quantification of the SNP alleles is of a great importance for practical use. Preexisting methods of quantifying SNPs, including real-time polymerase chain reaction (PCR) assays3 and DNA microarrays,4 often utilize a hybridization step between a single-stranded (ss) DNA analyte and an allele-specific oligodeoxyribonucleotide (ODN) probe. These methods take advantage of the fact that the thermodynamic stability of the fully matched double-stranded (ds) DNA between the target ssDNA and the allele-specific ODN probe is higher than that of the single-base-mismatched dsDNA between the nontarget, single-base-substituted ssDNA and the probe. However, because those methods were originally developed for discrimination of SNP alleles, they are not © XXXX American Chemical Society

Received: September 19, 2014 Accepted: October 30, 2014

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Scheme 1. Schematic Diagrams Illustrating Affinity Capillary Electrophoresis of the Wild-Type ssDNA Analyte (WT), the Single-Base-Mutated ssDNA Analyte (MT), and the Internal Standard ssDNA (IS) with a Random Sequence in the Presence of the PEG-b-ODN probe (a) in the Previous Study and (b) in the Present Studya

WT, MT, and IS are FITC-labeled, and detected with a fluorescent detector at the anode end of the tube. The closed gray circle represents a mononucleotide. The closed blue and red circles represent the SNP site of WT and MT, respectively. The open blue circle represents the mononucleotide complementary to the closed blue circle. The green wavy line represents the PEG segment. The expected electropherograms are also schematically depicted. a

GE Healthcare (Buckinghamshire, UK). The DNA concentration was determined by measuring the absorbance at 260 nm with a UV-2550 spectrophotometer from Shimadzu (Kyoto, Japan). Maleimide-terminated methoxy PEG with a nominal molecular weight of 20 000 (20K) or 30 000 (30K) was purchased from Nektar Therapeutics (San Carlos, CA) or NOF (Tokyo, Japan), respectively. Deionized water (>18.1 MΩ) purified with a Milli-Q instrument from Millipore (Billerica, MA) was used for all experiments except the PCR amplification. DNA Analytes. 5′-Fluorescein isothiocyanate (FITC)labeled 60-nucleotide (nt) DNA strands (WT1−WT4) and their single-base-substituted variants (MT1−MT4) were chemically synthesized and used as analytes. A 5′-FITC-labeled ssDNA with a random sequence (60 nt) was also synthesized and used as an internal standard (IS). All sequences are shown in Figure S1 (Supporting Information). The folded structure of the analyte under the electrophoresis conditions was calculated using an mfold web-based program.15 5′-FITC-labeled 71 nt ssDNA analytes termed WT5 and MT5 (Figure S1, Supporting Information) were prepared using PCR amplification of a fragment of the scytalone dehydratase gene of rice blast fungus and its single-base mutant. First, genomic DNA was extracted from Magnaporthe grisea mycelia by using a Nucleon PhytoPure kit (GE Healthcare). The fungi were the kind gift of Bayer CropScience KK Yuki Research Centre. Next, a 607 base-pair (bp) DNA fragment including the scytalone dehydratase gene was amplified from the genomic DNA by PCR (1st). The sequences of the amplified fragment and the primers are shown in Figure S2 (Supporting Information). After 30 cycles using steps of 94 °C for 30 s, 57 °C for 60 s, and 72 °C for 90 s with a thermal cycler (TGradient; Biometra, Göttingen, Germany), the reaction solution was kept at 4 °C. The PCR-amplified DNA fragment was then cloned into a pCR2.1-TOPO plasmid (3900 bp) by using a TOPO TA cloning kit from Invitrogen (Carlsbad, CA).

the WT peak. The separation of the MT and IS is an essential requirement for application of this affinity CE to SNP allele frequency estimation. This is because fluorescein isothiocyanate (FITC)-labeled PCR primers, which are used for preparing ssDNA analytes from real genetic samples, could contaminate the analyte solution. The mobility of the primers is almost identical to that of the IS, because the electrophoretic mobility of ssDNA in free-solution CE is independent of the sequence and length, provided that the base number is larger than 10.9 Previous works have demonstrated that simultaneous use of two different affinity probes enabled the desired separation of the WT, MT, and IS.10−12 However, further simplification and rationalization are expected to facilitate the practical use of this approach. In the current study, we separate the analytes using a single PEG-b-ODN probe (Scheme 1b). By optimizing the design of the probe’s ODN sequence, it should be possible to concurrently realize strong, weak, and no binding of the probe to the WT, MT, and IS, respectively. Therefore, we focus on establishing a guideline for selecting an appropriate ODN sequence in order to achieve such separation of the given analytes. Recently, we have found that use of PEG-b-PNA allowed clear separation of the WT, MT, and IS;13,14 however, the lack of databases of thermodynamic parameters involved in the heteroduplex formation between DNA and PNA poses obstacles to the rational design of the PNA sequence. Therefore, the use of a native DNA segment could have practical advantages for optimization of the base sequence.

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MATERIALS AND METHODS General. All reagents were purchased from Wako (Osaka, Japan) unless otherwise noted. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was obtained from Pierce (Rockford, IL). All chemically synthesized DNA strands were purchased from Tsukuba Oligo Service (Ibaraki, Japan). The 5′-thiolterminated DNA was purified through a NAP-5 column from B

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The resulting plasmid was amplified in Escherichia coli JM 109 and purified by using an RPM kit from Bio101 (Carlsbad, CA), followed by sequencing to confirm the cloned gene. Employing the cloned genes as a template, PCR (2nd) was performed with a 5′-FITC-labeled forward primer and a 5′-phosphorylated reverse primer. The sequences are shown in Figure S2 (Supporting Information). The PCR reactions were performed in 20 μL scale. Each reaction mixture was composed of 2 μL of approximately 5 nM dsDNA template, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 4 μL of 5× PrimeSTAR buffer (Mg2+ plus) (Takara Bio, Ohtsu, Japan), 1.6 μL of 2.5 mM of each dNTP mixture (Takara Bio), and 0.2 U of PrimeSTAR HS DNA polymerase (Takara Bio). Nuclease-free water of molecular biology grade from Sigma-Aldrich (St. Louis, MO) was used for preparing the reaction solutions. After 30 cycles using steps of 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 15 s, the reaction solution was kept at 4 °C. The PCR products were purified using a MinElute PCR purification kit from Qiagen (Hilden, Germany), and were identified with agarose gel electrophoresis; the mobility of the 71-bp PCR products was somewhat larger than that of a 100-bp size marker (Figure S3 in the Supporting Information). Then, the 5′phosphorylated antisense strands of the PCR products were digested by λ exonuclease in 20 μL scale. Each reaction mixture was composed of 10 μL of the PCR solutions, 5 U of λ exonuclease, and its reaction buffer (New England Biolabs Japan, Tokyo, Japan). The mixture was incubated at 37 °C for 1 h and at 75 °C for 10 min using the thermal cycler. After the digestion, the residual 5′-FITC-labeled sense strands (WT5/ MT5) were purified using a QIAquick nucleotide removal kit (Qiagen). Affinity Probes. The affinity probe, PEG-b-ODN(n), was synthesized by the previously reported method.6 A base number n of the ODN segment is shown in parentheses. The sequence of the ODN segments was designed to be complementary to part of the WT analyte including the SNP site (Figure S1, Supporting Information). The number-average molecular weight (Mn) and the molecular weight distribution (Mw/Mn) of the probes for WT1/MT1 were determined by aqueous size exclusion chromatography (Table S1 in the Supporting Information). The observed Mn values agreed fairly well with the calculated ones. In addition, all Mw/Mn values were lower than 1.2. These results indicated that the affinity probes were prepared successfully. 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 CEP-coated capillary tube (internal diameter, 75 μm; total length, 50.5 cm; effective length, 40.5 cm) with a precoated inner surface from Agilent Technologies (Wilmington, DE). This tube was used to suppress an electroosmotic flow. FITC-labeled analytes were detected with the LIF detector (excitation at 488 nm and recording emission at 520 nm) at the anode end of the capillary tube. Representative electrophoresis conditions were as follows. The capillary temperature was held constant at 60 °C with a recirculating liquid coolant system. As a running buffer, 50 mM Tris-borate buffer (pH 7.4) containing 10 mM NaCl and 0.5 mM MgCl2 was used. MgCl2 was employed to regulate the ionic strength of the running buffer, whereas 10 mM NaCl was added to meet the requirements of the theoretical calculations used here. Prior to electrophoresis, a solution of the affinity probe (5 μM) in the running buffer was injected into the capillary tube from the

cathode end by positive pressure (20 psi for 45 s). Next, a buffer solution of WT and MT (50 nM each) and IS (10−25 nM) was introduced into the capillary tube using a similar method (0.5 psi for 10 s). Electrophoresis was performed under reversed polarity with a constant voltage of −15 kV for 25 min. Between measurements, the capillary tube was sufficiently washed with deionized water (20 psi for 1 min) and then filled with the running buffer containing the affinity probe (20 psi for 1 min). Each CE was carried out at least in triplicate to confirm the reproducibility. A resolution parameter (RS) and a peak area ratio were used to evaluate the probe’s separating ability. RS was defined by R S = 2(t WT − tMT)/(WWT + WMT)

(1)

where tWT and tMT are the migration time of WT and MT, and WWT and WMT are the full widths of the WT and MT peaks, respectively. Similarly, the RS value was calculated for the separation between MT and IS. For determining the peak area ratio, each peak area was divided by its migration time to avoid changes in the peak area due to shifts in the migration time.16 The mobility analysis was performed with the reported method based on a Lineweaver−Burk-type plot.8 The electrophoretic mobility of the analyte (μ) was formulated as follows μ = {1/(1 + K[P])}μD + {K[P]/(1 + K[P])}μC

(2)

where K is the binding constant of the complex between the probe and the analyte, μD and μC are the electrophoretic mobility of free ssDNA and the complex, respectively, and [P] is the equilibrium concentration of the probe. Melting Temperature Measurement. The melting curve of folded ssDNA analyte (1 μM) was obtained by measuring the change of absorbance at 260 nm as a function of temperature with a UV-2550 spectrophotometer equipped with a TMSPC-8 temperature controller unit (Shimadzu). The heating and cooling ramp was 1 °C/min. The sample-holding 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. The DNA-concentration dependence of Tm for the duplex between the probe’s ODN segment and the analyte, which was truncated to make it equivalent in length to the ODN segment, was investigated to obtain the K value. Plots of Tm−1 versus log(CT/4), where CT is the total DNA strand concentration, were used to determine ΔH° and ΔS° from the slope and intercept according to the equation17 Tm−1 = 2.303R log(C T/4)/ΔH ° + ΔS°/ΔH °

(3)

with R being the gas constant. ΔG° and K were calculated from ΔH° and ΔS° by using the equations ΔG° = ΔH ° − T ΔS°

(4)

K = exp( −ΔG°/RT )

(5)

where T is the measurement temperature. The K values were also calculated by using the HyTher program.18

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RESULTS AND DISCUSSION Affinity Capillary Electrophoresis of the Folded DNA. As an analyte, we used a mixture of equal amounts of WT1 and MT1, which correspond to a fragment of the scytalone dehydratase gene of rice blast fungus and its single-base mutant at codon 75 (GTG to ATG), respectively. This point

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mutation confers resistance to a melanin biosynthesis inhibitor targeting scytalone dehydratase.19−21 To separate a mixture of WT1, MT1, and IS by free-solution capillary electrophoresis, we prepared PEG-b-ODN(20) as an affinity probe. The base sequence of the ODN segment was designed to be complementary to a part of WT1 including the SNP site (Figure 1a). We tentatively determined the base number of the

Figure 1. (a) Sequences of the ssDNA analytes (in part) and the ODN segments of the affinity probes. The analytes (WT1 and MT1) and the internal standard (IS) are FITC-labeled at the 5′-terminus. The SNP site is shown in bold. Total sequences of WT1, MT1, and IS are given in Figure S1 (Supporting Information). (b) Most stable secondary structure around the probe-targeting region of WT1 and MT1 predicted using mfold. A dashed line indicates base pairing. The calculated overall structures are shown in Figure S4a (Supporting Information).

Figure 2. Electropherograms showing the separation of WT1, MT1, and IS with the PEG-b-ODN(20) probe. Conditions: running buffer, 50 mM Tris-borate (pH 7.4) containing 10 mM NaCl and 0.5 mM MgCl2; [probe] = 5.0 μM; [WT1] = 50 nM; [MT1] = 50 nM; [IS] = 25 nM; capillary temperature, 30−60 °C; applied voltage, −15 kV.

ODN segment to be 20, which was considerably greater than the number used in our previous studies (7−10).6−8,10 This is because we tried to induce simultaneous strong/weak affinity interaction between WT1/MT1 and the probe, respectively (Scheme 1b). Figure 2 shows the electropherograms obtained at 30 °C−60 °C. The number of observed peaks differed among the three temperature ranges: (i) one peak was seen at 30 °C, (ii) two at 35 °C−55 °C, and (iii) three at 57.5 °C−60 °C. At 30 °C; we observed a single peak at 7 min. No retardation occurred for the WT1 peak, indicating that a negligibly weak interaction took place between WT1 and the probe, even though the probe’s ODN segment appeared to be long enough. This result was different from those of our previous studies;6−8,10 the ODN segment of 10 bases was long enough to cause the retardation. An explanation for this discrepancy is given below. When the temperature was increased to 35 °C−55 °C, the WT1 and MT1 peaks were concomitantly retarded. An affinity interaction took place between WT1 and the probe, albeit at higher temperature. In discrete runs, we confirmed that the retardations for WT1 and MT1 were basically identical (data not shown). When the temperature was further increased from 57.5 to 60 °C, the retardation of the MT1 peak was gradually diminished, while that of the WT1 peak was unchanged. As a consequence, the use of PEG-b-ODN(20) at 60 °C enabled the separation of WT1, MT1, and IS. Intramolecular base pairing accounts for the very peculiar electrophoretic behaviors of WT1 and MT1.We calculated the most stable secondary structure of the probe-binding region within WT1 and MT1 at 30 °C using mfold (Figure 1b).15 The probe-binding regions were involved in the hairpin structure. The Tm value was calculated to be 50 °C for WT1 and 51 °C for MT1 (Figure S4a, Supporting Information). Also, the Tm

value was experimentally determined to be 49 °C for WT1 and 45 °C for MT1 (Figure S4b). All of these values were much higher than the capillary temperature (30 °C). Thus, the folded analytes were incapable of hybridizing to the probe at 30 °C, with the result that there was no retardation for the peaks (Scheme 2a). When the temperature was increased to 35 °C− 55 °C, WT1 and MT1 were gradually unfolded to exhibit a random-coil structure; they were allowed to hybridize to the probe. Due to its long ODN segment, the PEG-b-ODN(20) formed not only a fully matched duplex with WT1 but also a single-base-mismatched duplex with MT1. The Tm value calculated with the HyTher program18 was 72 °C for the fully matched duplex and 66 °C for the mismatched one. Because their temperatures were significantly higher than the capillary temperature (35 °C−55 °C), each analyte formed a stable complex with the probe during electrophoresis. Consistently, the mobilities of WT1 and MT1 were almost identical (Scheme 2b). The details of the mobility analysis are given below. In the third temperature range of 57.5 °C−60 °C, the retardation of the MT1 peak from the IS peak became smaller than that of the WT1 peak. This is because the singlebase-mismatched complex between MT1 and the probe partially dissociated owing to the lower stability, while WT1 still formed a stable complex (Scheme 2c). As a result, a clear separation of WT1, MT1, and IS was achieved at 60 °C. A similar temperature dependence was observed for PEG-bODN(16) and PEG-b-ODN(18) (Figure S5, Supporting Information). Optimization of the Electrophoresis Conditions. Next, we optimized the base number of the probe and the salt D

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Scheme 2. Schematic Diagrams Illustrating Affinity Capillary Electrophoresis of WT1, MT1, and IS with PEG-b-ODN(20) at (a) 30 °C, (b) 35°C−55 °C, and (c) 57.5 °C and 60 °Ca

a

The obtained electropherograms are also schematically depicted.

Figure 3. (a) Electropherograms showing the separation of WT1, MT1, and IS with PEG-b-ODN(16), PEG-b-ODN(18), or PEG-b-ODN(20). Conditions: running buffer, 50 mM Tris-borate (pH 7.4) containing 10 mM NaCl and 0−1 mM MgCl2; [probe] = 5.0 μM; [WT1] = 50 nM; [MT1] = 50 nM; [IS] = 25 nM; capillary temperature, 60 °C; applied voltage, −15 kV. (b) Salt concentration dependence of resolution parameters (RS) for the separation of MT1 and IS (open circles) and the separation of WT1 and MT1 (closed circles). Black solid lines are drawn as a guide. Red dashed lines indicate the RS value of 1.5.

concentration of the running buffer at 60 °C. Previous studies have already demonstrated that increasing the base number of the probe and the salt concentration can enhance the thermodynamic stability of the affinity complex, resulting in

greater retardation of the corresponding peak.6−8,10 The same held true for the electropherograms obtained here (Figure 3a). When PEG-b-ODN(16) was used, the WT1 peak was gradually retarded with an increase in the MgCl2 concentration from 0 to E

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the fully matched complex between PEG-b-ODN(20) and WT1 was almost identical to that for the single-basemismatched complex between the probe and MT1, suggesting that the single-base mismatch caused the negligibly small difference in hydrodynamic friction that the complex received during electrophoresis. The ssDNA analyte existed as a complex in a part of the migration time and did as a free form in the rest of the total migration time. Submitting the Kelps value into the coefficient of the second term in eq 2 gave the fraction of migration time for the complex:5 99% for WT1 and 54% for MT1. Therefore, WT1 migrated as a complex for almost all of the migration time, while MT1 migrated as a complex for half of the total migration time and as a free ssDNA for the other half. The validity of this mobility analysis was verified as follows. We also determined the binding constant (Kmelt) using the Tm values observed for the fully matched duplex between the pristine 20 nt ODN segment and the WT1 (or MT1) truncated to be of the same length (20 nt) (Table S2 in the Supporting Information). Using eqs 3−5, we calculated the Kmelt values for WT1 and MT1. These values agreed well with the corresponding Kelps values (Table 1). Guidelines for the Sequence Design of an Affinity Probe. We found four other conditions to achieve clear separation of WT1, MT1, and IS. Figure 3b shows that RS1 and RS2 values larger than 1.5 were also obtained using PEG-bODN(18) with 1.0 mM MgCl2 at 60 °C, or using PEG-bODN(20) with 0.75 mM MgCl2 at 60 °C. In addition, we were able to separate them using PEG-b-ODN(16) with 0.5 mM MgCl2 at 50 °C (Figure S5, Supporting Information) and using PEG-b-ODN(18) with 0.5 mM MgCl2 at 55 °C (data not

1.0 mM; however, no retardation of the MT1 peak was observed. In contrast, when using PEG-b-ODN(18) with 1.0 mM MgCl2, we observed small and large retardations of the MT1 and WT1 peaks, respectively. When using PEG-bODN(20), we also achieved a clear separation with 0.5, 0.75, and 1.0 mM MgCl2. To quantitatively assess the probe’s separation performance, we calculated RS1 for the IS and MT1 peaks, and RS2 for the MT1 and WT1 peaks (Figure 3b). It is generally accepted that RS > 1.5 is necessary to achieve the baseline separation.22 Both RS1 and RS2 became sufficiently larger than 1.5 when using PEG-b-ODN(20) with 0.5 mM MgCl2. Under the optimized conditions, we determined the binding constant (Kelps) and the electrophoretic mobility (μC) of the complex between the analyte and the probe using a Lineweaver−Burk-type analysis (Table 1).8 The μC value for Table 1. Electrophoretic Mobility (μC) and Binding Constant (K) of the Complex between the Probe and the Analyte at 60 °C probe/analyte

μCa (cm2/(V·s))

Kelpsa (M−1)

Kmeltb (M−1)

PEG-b-ODN(20)/WT1 PEG-b-ODN(20)/MT1

−2.1 × 10−4 −1.9 × 10−4

1.8 × 107 2.3 × 105

6.5 × 107 3.5 × 105

a

Determined by the electrophoretic mobility analysis in 50 mM Trisborate buffer (pH 7.4) containing 10 mM NaCl and 0.5 mM MgCl2. b Determined from the Tm values for the DNA duplex between the unmodified ODN (20 bases) and the truncated analyte (20 bases) at the same salt concentrations.

Figure 4. (a) Electropherograms showing the separation of a mixture of WT2, MT2a, and IS (left), a mixture of WT2, MT2t, and IS (center) or a mixture of WT2, MT2c, and IS (right) with PEG-b-ODN(26) and 0.5 mM MgCl2 at 53 °C. (b) Electropherogram showing the unsuccessful separation of WT2 and MT2t with PEG-b-ODN(26) and 0.5 mM MgCl2 at 50 °C. (c) Electropherogram showing the separation of WT3, MT3, and IS with PEG-b-ODN(14) and 0.5 mM MgCl2 at 30 °C. (d) Electropherogram showing the separation of WT4, MT4, and IS with PEG(30K)-bODN(18) and 0.5 mM MgCl2 at 55 °C. Conditions: running buffer, 50 mM Tris-borate (pH 7.4) containing 10 mM NaCl; [probe] = 5.0 μM; [WT] = 50 nM; [MT] = 50 nM; [IS] = 10 nM (c) or 25 nM (a, b, and d); applied voltage, −15 kV. F

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than 106 M−1, no separation of MT2t and WT2 was observed due to the overly strong affinity (Figure 4b). Second, we used a fragment of the mitochondrial cytochrome b codon 133−153 of cucumber powdery mildew and its single-base mutant at codon 143 (GGT to GCT). The single-base substitutions induce strobilurin-related fungicide resistance.24 The sequences of the analytes termed WT3 and MT3 are shown in Figure S1 (Supporting Information). We measured Tm for the folded structure with 10 mM NaCl and 0.5 mM MgCl2 (Table S5 in the Supporting Information). We set the capillary temperature at 30 °C, which was higher than the observed Tm values. We then calculated the Kcalc value for the duplex between the unmodified ODN (12, 14, or 16 nt) and the truncated MT3 at 30 °C. Because only the ODN(14) followed the guideline, we synthesized PEG-b-ODN(14) for the probe. Figure 4c shows that MT3 was separated from WT3 and IS. Third, we selected a fragment of the same mitochondrial cytochrome b gene of wheat powdery mildew and its singlebase mutant. The sequences of the analytes termed WT4 and MT4 are shown in Figure S1 (Supporting Information). To confirm the generalizability of the guideline, we altered the PEG length from 20K to 30K. Previous studies revealed the effect of this parameter on the electrophoretic mobility of the target analyte;10 increasing the PEG length enhances the hydrodynamic friction exerted on the complex during electrophoresis, leading to a decrease in the absolute μC value. As a result, the mobility of the target ssDNA is reduced. We experimentally determined Tm for the folded structure with 10 mM NaCl and 0.5 mM MgCl2 to be 48 °C for WT4 and 27 °C for MT4 (Table S6 in the Supporting Information). We set the capillary temperature at 55 °C (>48 °C). Then, we determined Kcalc for the duplex between the unmodified ODN (16, 18, or 20 nt) and the truncated MT4 at 55 °C. Because only the ODN(18) followed the guideline, we synthesized PEG(30K)-bODN(18) using the same method. The electropherogram is depicted in Figure 4d, and it can be seen that the mixture of WT4, MT4, and IS was clearly separated. Applications to Quantitative SNP Analysis. Finally, we performed quantitative SNP analysis with the present method. We prepared 5′-FITC-labeled 71 nt ssDNA analytes termed WT5 and MT5 using PCR amplification of a fragment of the scytalone dehydratase gene of rice blast fungus and its singlebase mutant. We first extracted genomic DNA from Magnaporthe grisea mycelia for use in the PCR templates (Figure S6a in the Supporting Information). Next, a DNA fragment (607 bp) including the scytalone dehydratase gene was amplified by PCR (1st) using the extracted genomic DNA as a template. The obtained dsDNA fragment was then cloned into a plasmid, which was further amplified in E. coli. We mixed the plasmids incorporating the wild-type gene (Plasmid-WT) and the single-base-mutated one (Plasmid-MT) at a predetermined feed ratio. Subsequently, we performed the PCR (2nd) employing the cloned plasmids as a template with a 5′-FITC-labeled forward primer and a 5′-phosphorylated reverse primer (Figure S2, Supporting Information). Finally, we subjected the PCR products to enzymatic digestion of the 5′-phosphorylated antisense strand by λ exonuclease to yield WT5 and MT5. We conducted affinity CE of WT5/MT5 using PEG-bODN(18) with 10 mM NaCl and 0.5 mM MgCl2 at 55 °C, one of the optimal conditions established earlier (Table S3, Supporting Information). Figure 5 shows the electrophero-

shown). All favorable electrophoresis conditions are listed in Table S3 (Supporting Information). Using the HyTher program,18 we determined the binding constant (Kcalc) for the fully matched duplex between the ODN segment and the truncated WT1 under these conditions to be 2.1−17 × 108 M−1. Similarly, Kcalc for the single-base-mismatched duplex between the ODN segment and the truncated MT1 was 1.6− 13 × 106 M−1. On the basis of these results, we set guidelines for the design of an applicable ODN sequence. For an ssDNA analyte with a given sequence, the ODN sequence should be determined in such a way that (i) Kcalc for the fully matched duplex between the ODN and the truncated WT is ≥108 M−1 and (ii) Kcalc for the mismatched duplex between the ODN and the truncated MT is on the order of 106 M−1. Because the ODN sequence that follows the guideline (ii) ordinarily satisfies (i) as well, the guideline (ii) is hereafter considered solely. Next, we confirmed that WT1 and MT1 could not be successfully separated by using a probe designed outside of this guideline. As mentioned above, the Tm for the folded structure with 10 mM NaCl and 0.5 mM MgCl2 was determined to be 49 °C for WT1 and 45 °C for MT1. Therefore, we set the capillary temperature at 60 °C, which was higher than 49 °C, to avoid the undesirable self-folding of the analytes. We then determined Kcalc for the duplex between the ODN(16) and the truncated MT1 at 60 °C to be 3.0 × 104 M−1. Because this value was lower than 106 M−1, we observed no separation of IS and MT1 due to the weak affinity (Figure 3a, the leftmost column and the third row). Also, when setting the capillary temperature at 55 °C, the Kcalc value between the ODN(20) and the truncated MT1 at 55 °C was 1.6 × 108 M−1. This was higher than 106 M−1, and thus there was no separation of MT1 and WT1 due to the overly strong affinity (Figure 2, third row from the bottom). Examples of the Sequence Design of an Affinity Probe. According to the above guideline, we tried to predict appropriate ODN sequences for three different analytes. First, we chose a fragment of the DNA polymerase gene of the hepatitis B virus (HBV) and its single-base-substituted variants at codon 552 (ATG to ATA, ATT, or ATC) for analytes. The single-base mutations (M552I mutations) are related to the development of lamivudene-resistant HBV.23 The sequences of the analytes termed WT2, MT2a, MT2t, and MT2c are shown in Figure S1 (Supporting Information). We determined the Tm values for folding of the analytes from the melting curves to be 28 °C−33 °C with 10 mM NaCl and 0.5 mM MgCl2 (Table S4 in the Supporting Information). We decided to set the capillary temperature at 53 °C, which was far beyond the highest Tm value for the folding (33 °C). Next, we determined Kcalc for the DNA duplex between the ODN segment (24, 26, or 28 nt) and the truncated analyte at 53 °C using the HyTher program.18 The sequence is depicted in Figure S1 (Supporting Information). Because only the ODN(26) followed the guideline, we synthesized PEG-b-ODN(26). The electropherograms are shown in Figure 4a. Each of MT2a, MT2t, and MT2c was separated from WT2 and IS. Conversely, we observed that the separation of WT2 and MT2t was unsuccessful when using a probe designed outside of the guideline. When we set the capillary temperature at 50 °C, which was higher than the Tm values for the self-folding of WT2/MT2t, we obtained a Kcalc value of 5.3 × 107 M−1 for the duplex between the ODN(26) and the truncated MT2t at 50 °C. Because this value was higher G

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0.995 (Figure S6b, Supporting Information). These results strongly suggest that the present method could be of practical use for SNP allele frequency estimation.

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CONCLUSION In this study, we have set a guideline for the design of an affinity CE probe for separating WT and MT with a given base sequence. Specifically, the procedures to determine the ODN sequence of PEG-b-ODN can be summarized as follows. (1) Tm for folding of the ssDNA analyte is experimentally determined under the electrophoresis conditions. This step might be replaced by prediction using mfold with an improved accuracy. (2) The capillary temperature T is set to be above the Tm value to unwind the folded analytes during electrophoresis. (3) The binding constant K of the DNA duplex between a tentative ODN segment and the analyte at the capillary temperature T is calculated with HyTher. The ODN sequence is optimized in such a way that the Kcalc value for the mismatched duplex between the ODN segment and MT is on the order of 106 M−1. In general, the chemical modification of biomolecules with synthetic polymer chains can significantly influence their physicochemical properties and biochemical activities. Therefore, a small undesired side effect of PEGylation on the thermodynamics of the original DNA hybridization should be, at least partially, responsible for the present accurate prediction. The affinity CE with PEG-b-ODN thus designed is useful for quantifying SNP alleles. This method could be of general applicability to bioanalysis and medical diagnosis, such as for evaluating the occurrence rate for fungicide-resistant pathogens or determining a ratio between normal and tumor cells within given tissue samples.

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Figure 5. Electropherograms showing the separation of WT5, MT5, and contaminants (CT) with PEG-b-ODN(18) and 0.5 mM MgCl2 at 55 °C. For preparing WT5 and MT5 by PCR, the mixtures of PlasmidWT and Plasmid-MT were used for a template at different feed ratios. Conditions: running buffer, 50 mM Tris-borate (pH 7.4) containing 10 mM NaCl; [probe] = 5.0 μM; applied voltage, −15 kV. The peak area ratios expressed are the means of triplicate measurements (±SD).

ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*T. Takarada. E-mail: [email protected].

grams. Three distinct peaks appeared: a fast one at 5 min for contaminants (CT), an intermediate one at 7 min for MT5, and a slow one at 10 min for WT5. The migration time of CT agreed well with that of IS (data not shown). We assigned CT to the FITC-labeled forward primers and/or the FITC-labeled dsDNA that were left undigested by λ exonuclease, either or both of which could have been incompletely excluded from the final ssDNA products. The peak of the PCR product without the enzymatic digestion was actually observed at 5 min (data not shown). The discrimination of MT5 from CT was necessary to precisely quantify the ratio of MT5. As expected, the present affinity CE allowed quantitative SNP analysis based on the peak area ratio. The averaged value of the peak area ratios between WT5 and MT5 that were obtained by the three experiments agreed well with the value of the feed ratio between Plasmid-WT and Plasmid-MT (Figure 5). The averaged difference between the feed ratio and the observed peak area ratio, referred to as accuracy, was determined to be 3.1%. This accuracy is relatively favorable compared to those of preexisting methods using an allelespecific ODN probe, which range from 0.23 to 6.7%.11,14,25 A good linear relationship between the observed and feed ratios yielded the regression line with a correlation coefficient of

Present Address §

Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant for Ecomolecular Science Research provided by RIKEN and by a Grant-in-Aid for Scientific Research (A) (No. 20245020) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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