Small-Molecule Binding at an Abasic Site of DNA: Strong Binding of

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Small-Molecule Binding at an Abasic Site of DNA: Strong Binding of Lumiflavin for Improved Recognition of Thymine-Related Single Nucleotide Polymorphisms N. B. Sankaran,†,‡,§ Yusuke Sato,† Fuyuki Sato,†,¶ Burki Rajendar,† Kotaro Morita,† Takehiro Seino,† Seiichi Nishizawa,†,‡ and Norio Teramae*,†,‡ Department of Chemistry, Graduate School of Science, Tohoku UniVersity, Aoba-ku, Sendai 980-8578, Japan, and CREST, Japan Science and Technology Agency (JST), Sendai 980-8578, Japan ReceiVed: September 27, 2008; ReVised Manuscript ReceiVed: NoVember 24, 2008

The binding behavior of lumiflavin, a biologically vital ligand, with DNA duplexes containing an abasic (AP) site and various target nucleobases opposite the AP site is studied. Lumiflavin binds selectively to thymine (T) opposite the AP site in a DNA duplex over other nucleobases. Using 1H NMR spectroscopy and fluorescence measurements, we show that ligand-DNA complexation takes place by hydrogen-bond formation between the ligand and the target nucleobases and by stacking interactions between the ligand and the nucleobases flanking the AP site. From isothermal titration calorimetric experiments, we find that ligand incorporation into the AP sites is primarily enthalpy-driven. Examination of ionic strength dependency of ligand binding with DNA reveals that ligand-DNA complexation is a manifestation of both electrostatic and nonelectrostatic interactions and that the contribution from the nonelectrolyte effect is fundamental for the stabilization of the ligand-DNA complex. In comparison to riboflavin, reported previously as a T-selective ligand, lumiflavin binds to the DNA much more strongly and is a more promising ligand for efficient detection of T-related single nucleotide polymorphisms. 1. Introduction During the last several decades, a lot of interest has been paid to DNA-binding molecules. There has been particular interest regarding understanding the nature of smallmolecule-nucleic acids interactions since DNA, with its role in genetic diseases, is an exciting target for drug design in pharmaceuticals.1 Any alterations to the unique structure of DNA will lead to pronounced changes in its structural and functional properties. The manifestation of the structure of DNA by small molecules has led to identifying many DNA-binding molecules that act as potential agents in drug design and therapeutics.2,3 Interactions of molecules with DNA are highly sequencespecific, wherein the interacting molecule targets a particular nucleobase in the DNA sequence. Most of the DNA-binding ligands are either groove binders4-9 or intercalators;10-13 classical examples belonging to the first class include lexitropsins4 and bis(amidinium) compounds,5 and those belonging to the latter class include daunomycin,10 noglamycin,11,12 ethidium bromide,13 and propidium iodide.13 A thorough knowledge of the structure of ligand-DNA complexes and the thermodynamics of their formation helps researchers in manipulating the parameters to enhance the ligand-DNA binding affinities and to improve the base sequence specificity. Related to the DNA-binding ligands and DNA lesions, Nakatani and co-workers reported specific recognition of a single guanine bulge by 2-acrylamino-1,8-naphthyridines,14 and they have successfully applied the binding between naphthyridine * To whom correspondence should be addressed. Tel: +81-22-795-6549. Fax: +81-22-795-6552. E-mail: [email protected]. † Tohoku University. ‡ CREST-JST. § Present address: Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal, QC H3A 2K6, Canada. ¶ Present address: Department of Chemistry, Faculty of Science and Technology, Sophia University, Chiyoda-ku, Tokyo 102-8554, Japan.

derivatives and the bulge sites in a DNA duplex to the selective detection of the mismatched base pairs,15-18 SNPs,15 and trinucleotide repeats19 based on surface plasmon resonance. We have also been engaged in developing new methods for typing of single nucleotide polymorphisms (SNPs) which are the genetic variations associated with various common diseases. In contrast to currently available methods20-24 that either require several time-consuming steps or use of several kinds of fluorophore-labeled oligodeoxynucleotides, and/or special enzymes, we proposed a new method of ligand-based fluorescence assay for their typing in combination with abasic (AP) sitecontaining DNA duplexes.25 Whereas AP sites are naturally formed in DNA,26,27 in our experiments, the AP site is artificially constructed by hybridizing two complementary single-strand oligo DNAs, one containing the AP site and the other carrying the nucleobase of our interest, hereafter called the target nucleobase, so that the target nucleobase is situated opposite the AP site after hybridization (Figure 1). The AP sites thus created in DNA duplexes are hydrophobic in nature. It can be expected that hydrophobic ligands will easily be able to enter the AP site and bind to the target nucleobase by hydrogen bonding with the target nucleobase and stacking with the nucleobases flanking the AP site. We earlier employed this design logic for nucleobase recognition using small hydrogen-bonding ligands such as naphthyridine derivatives,25,28,29 pteridine derivatives,30-32 pyrazine derivatives,33 alloxazine,34 and flavin derivatives.35-38 Naphthyridine25,28,29 and pteridine30,31 derivatives were respectively found to detect single-base mutations related to cytosine and guanine. Amiloride, one of the pyrazine derivatives, showed selective fluorescence response upon binding with thymine opposite an AP site in a DNA duplex (K11 ) 6.7 × 106 M-1 at 278 K).33 Alloxazine bound to adenine selectively over other nucleobases opposite the AP site (K11 ) 1.2 × 106 M-1 at 278 K).34 As for flavin derivatives, riboflavin (Chart 1) strongly

10.1021/jp808576t CCC: $40.75  2009 American Chemical Society Published on Web 01/09/2009

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J. Phys. Chem. B, Vol. 113, No. 5, 2009 1523 2. Experimental Section

Figure 1. Illustration of the structure of an AP-site-containing DNA duplex. The white strand represents the AP-site-containing DNA strand, and the gray strand represents the complementary strand with a target nucleobase.

CHART 1

bound to the DNA with target thymine (K11 ) 1.8 × 106 M-1 at 278 K) when incorporated into the AP sites.35 Other than their utilization for detection of thymine-related single-base mutations,35 such binding events had also been instrumental in the development of AP-site-based DNA aptamers for riboflavin.36 However, for practical applications, it is highly desired that ligands bind to the DNA more strongly. Our continued quest for stronger DNA-binding molecules has identified lumiflavin (Chart 1) as a promising candidate for improved detection of thymine-related single-base mutations. Lumiflavin, a biologically vital compound involved in the mechanism of liver uptake of riboflavin by human-derived Hep G2 cells,39,40 is found to bind to AP-site-containing DNA with significant strength; for example, the binding constant reaches 2.1 × 107 M-1 at 278 K when bound to DNA containing the thymine target. In comparison to the earlier report for riboflavin,35 the binding efficiency of lumiflavin is markedly improved, enabling it to be a more suitable ligand than riboflavin for detecting thyminerelated single-base mutations. In the present study, we discuss the binding efficiency of lumiflavin with DNA containing various target nucleobases opposite the AP site. The nature of nucleobase recognition is discussed using 1H NMR spectroscopy and fluorescence measurements. The thermodynamics of ligand-DNA complexation are examined using isothermal titration calorimetric experiments. Analysis of the thermodynamic data reveals that (1), as is typical of DNA intercalators, flavin incorporation into the AP sites is primarily driven by enthalpy changes and (2) nonelectrostatic effects are fundamental to the stabilization of flavin-DNA complexes.

Flavins were obtained from Aldrich Chemical Co., (Milwaukee, WI) and used as-received. The oligodeoxynucleotides used were custom-synthesized and HPLC-purified (>97%) by Nihon Gene Research Laboratories, Inc. (Sendai, Japan). For the synthesis of AP-site-containing DNAs, a propylene residue (spacer phosphoramidite C3, Spacer C3) was utilized. The concentrations of DNAs were estimated from the molar extinction coefficients at 260 nm:41 97 800 cm-1 M-1 for 5′-d(TCC AGX GCA AC)-3′; 106 080 cm-1 M-1 for 5′-d(GTT GCG CTG GA)-3′; 103 560 cm-1 M-1 for 5′-d(GTT GCC CTG GA)-3′; 108 420 cm-1 M-1 for 5′-d(GTT GCA CTG GA)-3′; and 104 000 cm-1 M-1 for 5′-d(GTT GCT CTG GA)-3′. Water was deionized (g18.0 MΩ cm specific resistance) using an Elix 5 UV water purification system and a Milli-Q Synthesis A10 system (Millipore Corp., Bedford, MA). Other reagents were of commercially available analytical grade and used without further purification. Unless otherwise stated, all measurements were carried out in 10 mM sodium cacodylate buffer solutions (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. The DNA solutions were annealed before measurements by heating the solutions at 75 °C for 10 min and then gradually cooling them down to 5 °C (3 °C/min). The DNA duplexes used for various experiments were prepared by hybridizing an 11-meric AP-site-containing DNA strand (5′-d(TCC AGX GCA AC)-3′; X is Spacer C3) with four kinds of complementary sequences having different target nucleobases (5′-d(GTT GCN CTG GA)-3′; N ) G, A, C, T). Thermal denaturation, fluorescence, and NMR measurements were carried out as described previously.36 NMR spectra were recorded with an AVANCE 600 spectrometer (600 MHz, Bruker) employing the WATERGATE method.42,43 For NMR measurements, DNA solutions were prepared in a Tris buffer solution (10 mM; pH 8.3) containing 100 mM NaCl and 1.0 mM EDTA. Isothermal titration calorimetric (ITC) measurements were carried out on a VP-ITC microcalorimeter interfaced to a Gateway PC for data acquisition (Microcal Corporation, Northampton, MA). Origin (version 7.0) software was used to analyze the data. Ligand solution (1.4 mL) in the sample cell was titrated using 10 µL volumes (first injection: 2 µL for 4 s; then 24 injections of 20 s duration at 4 min time intervals) of an 11-meric DNA duplex (5′-d(TCC AGX GCA AC)-3′/3′d(AGG TCT CGT TG)-5′; X is Spacer C3). As a control experiment, the solution without the ligand in the sample cell was titrated to obtain the dilution heat. The area of each injection peak was automatically determined. A binding isotherm was obtained when the total heat per injection (kcal mol-1 of injectant) was plotted against the molar ratio of the ligand to the DNA duplex. Control experiments were performed by titrating the oligo solutions against a solution of 10 mM sodium cacodylate buffer containing 100 mM NaCl and 1 mM EDTA. For the measurements of the specific heat capacity, the measurement temperatures were varied from 5 to 15 °C at intervals of 2.5 °C. 3. Results 3.1. Thermal Denaturation Studies. As an AP site, we have used a tetrahydrofuranyl residue (dSpacer)25,28,30,31 and a propylene residue (spacer phosphoramidite C3, Spacer C3)31-38 to examine the binding behaviors between fluorescent ligands and nucleobases opposite the AP site in DNA duplexes. Since pterin showed the stronger affinity with guanine for Spacer C3 than for dSpacer,31 Spacer C3 was used as an AP site in this study.

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TABLE 1: Melting Temperatures (Tm/°C) for the Binding of Lumiflavin with AP-Site-Containing DNA Having Various Target Nucleobasesa target base

Tm(-)

Tm(+) (∆Tm)

G A C T

34.3 ( 0.4 34.3 ( 0.4 31.8 ( 0.5 29.3 ( 0.4

34.7 ( 0.3 (+0.4) 35.0 ( 0.2 (+0.7) 35.3 ( 0.1 (+3.5) 36.3 ( 0.7 (+7.0)

a The sequence of the DNA duplex was 5-d(TCC AGX GCA AC)-3/3′-d(AGG TCN CGT TG)-5′, where X ) Spacer C3 and N ) G, A, C, T. [Sodium cacodylate buffer] ) 10 mM; [NaCl] ) 100 mM; [EDTA] ) 1 mM; pH ) 7.0; Tm(-) ) melting temperature of ligand-free DNA; Tm(+) ) melting temperature of ligand-added DNA; ∆Tm ) Tm(+) - Tm(-); [lumiflavin] ) 15 µM; [DNA duplex] ) 30 µM. Errors denote the standard deviation obtained from three repeated independent measurements.

First, we performed thermal denaturation experiments monitored by UV absorption to examine the binding behavior of lumiflavin with the AP-site-containing DNA duplexes having various target nucleobases. 11-Meric single-stranded DNA containing the AP site (5′-d(TCC AGX GCA AC)-3′; X is Spacer C3) was hybridized with four kinds of complementary sequences, one each with target bases guanine, adenine, cytosine, and thymine (5′-d(GTT GCN CTG GA)-3′; N ) G, A, C, T). The change in absorbance of these hybridized DNA duplexes at 260 nm in the absence and in the presence of lumiflavin was monitored as a function of temperature (20-85 °C) to obtain the melting temperature (Tm). The resulting melting temperature profile was differentiated to determine the Tm values (Table 1). As seen in Table 1, in the absence of lumiflavin, Tm values are low for pyrimidine bases opposite the AP site, in contrast to purine bases. This difference in Tm values can be ascribed to the structure of a DNA duplex containing an AP site. It has been reported that a purine base tends to be stacked inside of the DNA helix or in a dynamic equilibrium between an intra- and extrahelical conformation when the purine base faces the AP site and that a pyrimidine base tends to be in an extrahelical conformation or in a dynamic equilibrium between an intraand extrahelical conformation when the pyrimidine base faces the AP site.44,45 These structural features of a DNA duplex containing an AP site determine the Tm values of purine or pyrimidine base opposite the AP site in the absence of lumiflavin. Upon adding lumiflavin to the DNA duplex, an increase in the Tm value (∆Tm) is observed, indicating an enhanced stability of the ligand-complexed DNA. As listed in Table 1, an increase in the Tm value up to 7.0 °C was observed when lumiflavin made complexation with the AP-site-containing DNA with target thymine. With cytosine opposite the AP site, the Tm value of lumiflavin-incorporated DNA was increased by 3.5 °C. ∆Tm values for ligand-incorporated DNA with targets adenine or guanine were, however, significantly less. 3.2. Fluorescence Studies. As for fluorescence studies on AP sites, fluorescent probes, such as 2-aminopurine and a coumarin dye, sensitive to the microenvironment, have been used to examine the polarity and stacking efficiency at the AP sitebysteady-state45,46 andtime-resolvedfluorescencemeasurements.47-49 In contrast to these studies, we have studied fluorescent ligands to discriminate nucleobases opposite the AP site in DNA duplexes. Here, the binding efficiency of lumiflavin with APsite-containing DNA duplexes (5′-d(TCC AGX GCA AC)-3′/ 3′-d(AGG TCN CGT TG)-5′; X is Spacer C3; N) G, A, C, T) was examined by fluorescence experiments. As shown in Figure 2, lumiflavin shows significant quenching of its fluorescence upon addition of DNA duplexes containing an AP site, indicat-

Figure 2. Fluorescence spectra of lumiflavin without and with APsite-containing DNA duplexes (5′-d(TCC AGX GCA AC)-3′/3′-d(AGG TCN CGT TG)-5′, where X ) Spacer C3 and N ) G, A, C, T). [Sodium cacodylate buffer] ) 10 mM; [NaCl] ) 100 mM; [EDTA] ) 1 mM; [lumiflavin] ) 15 µM; [DNA] ) 30 µM; λex ) 474 nm; temperature ) 293 K; pH ) 7.0.

Figure 3. Fluorescence titration curves for lumiflavin with AP-sitecontaining DNA duplexes (5′-d(TCC AGX GCA AC)-3′/3′-d(AGG TCN CGT TG)-5′, where X ) Spacer C3 and N ) G, A, C, T). [Sodium cacodylate buffer] ) 10 mM; [NaCl] ) 100 mM; [EDTA] ) 1 mM; [lumiflavin] ) 10 µM; λex ) 474 nm; λem ) 520 nm; temperature ) 293 K; pH ) 7.0. F and F0 denote the fluorescence intensities of lumiflavin in the presence and absence of DNA, respectively.

ing incorporation of the ligand into the AP site. The degree of quenching depends on the target nucleobase opposite the AP site, and the strongest quenching is observed for target thymine followed by cytosine. For adenine and guanine opposite the AP site, the fluorescence quenching is moderate. The binding behaviors of lumiflavin were further examined by fluorescence titration experiments, and the results are shown in Figure 3. The fluorescence titration curves were best fit to the 1:1 stoichiometry, and the estimated 1:1 binding constants (K11) between lumiflavin and the DNA duplexes are summarized in Table 2. From Table 2, it is clear that the K11 value depends on the target nucleobases opposite the AP site. A higher K11 value is obtained when thymine is situated opposite the AP site, followed by cytosine. With other target nucleobases, K11 values are substantially less. In addition, considerable increase in K11 values was observed at low temperatures (Figure S1, Supporting Information). In comparison with a K11 of 1.2 × 106 M-1 at 293 K for the binding of lumiflavin with DNA having target thymine (Table 2), K11 reached 2.1 × 107 M-1 when titrated at 278 K. For DNA duplexes having other target nucleobases, an increase in K11 values was also observed with decreasing temperature. 3.3. Ionic Strength Dependency of Flavin-DNA Binding. The ionic strength dependency of ligand-DNA binding affinities can be used to obtain the polyelectrolyte and nonelectrostatic components of the overall binding free energy of ligand-DNA

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TABLE 2: Binding Constantsa (K11, M-1) for the Complexation of Lumiflavin with AP-Site-Containing DNA Having Various Target Nucleobases target base K11 a

G

A

(7.1 ( 0.5) × 10

4

b

C

(7.1 ( 0.6) × 10

4

T

(3.9 ( 0.6) × 10

5

(1.2 ( 0.1) × 106

From fluorescence titration experiments at 293 K; [lumiflavin] ) 10 µM; λex ) 474 nm; λem ) 520 nm. b Errors denote the fitting error.

interactions.50 The effect of ionic strength on the binding efficiency of lumiflavin with AP-site-containing DNA having a thymine target was examined by fluorescence titration experiments at different salt concentrations. The molar concentrations of NaCl used were 0.11, 0.16, 0.21, 0.26, 0.31, 0.36, 0.41, and 0.51. K11 values obtained from fluorescence titration experiments under these conditions were plotted against the salt concentrations (Figure 4). The charge on the ligand when binding to the DNA was then calculated using the following equation50

S)

δ ln K11 δ ln[Na+]

) -ZΨ

(1)

where S is the slope of the linear least-squares analysis of the ln K11 versus ln[Na+] plot, Z is the number of counterions released per ligand binding, which is equivalent to the apparent charge of the ligand, and Ψ is a constant equal to the fraction of counterions associated per phosphate (0.88 for B-type DNA44). The apparent charge on lumiflavin when binding to DNA with target thymine was calculated as 0.45 using the above equation. The contribution of electrostatic interactions toward the total free energy was then calculated using the following equation51

∆Gpe ) -SRT ln[Na+]

(2)

where R is the gas constant and T is the absolute temperature. The polyelectrolyte (∆Gpe) and nonelectrostatic (∆Gt) contributions were examined from the overall binding free energy using the following expression

∆G ) ∆Gpe + ∆Gt

d(TCC AGX GCA AC)-3′/(3′-d(AGG TCT CGT TG)-5′; X is Spacer C3) was injected into a constant volume (1.4 mL) of the ligand in the sample cell. The data obtained from ITC experiments at 278 K for the interaction of flavins with APsite-containing DNA duplexes having target thymine base are summarized in Table 3. The corresponding binding isotherms are shown in Figure 5. It can be seen from Table 3 that flavins bind to the DNA with a 1:1 stoichiometry and that such binding events are enthalpy-driven. 4. Discussion 4.1. On the Binding Efficiency of Flavins at the AP Site. As can be seen from Table 1, in solutions buffered to pH 7.0 (I ) 0.11 M), the increase in melting temperature (∆Tm) for the AP-site-containing DNA duplexes is larger when lumiflavin binds to the DNA having the thymine target. ∆Tm is significantly less when the target nucleobase is cytosine. With adenine and guanine targets, ∆Tm becomes negligible. ∆Tm values for lumiflavin-DNA binding are significantly higher when compared to those for the binding of riboflavin with 11-mer APsite-containing DNA of identical sequences, reported previously.35 For comparison, we obtain the ∆Tm value for lumiflavinincorporated 11-meric AP-site-containing DNA with thymine target of 7.0 °C, whereas for the complexation of riboflavin with a DNA duplex of identical sequence, ∆Tm was 4 °C,35 indicating a stronger binding efficiency of lumiflavin over riboflavin with an identical DNA sequence. For AP-site-containing DNA with cytosine target, ∆Tm values were respectively found to be 3.5 and 2 °C after the incorporation of lumiflavin and riboflavin35 into the AP sites. For any of the target nucleobases opposite

(3)

From equation 2, the polyelectrolyte contribution toward the total binding free energy for the binding of lumiflavin with DNA having target thymine was calculated as -0.47 kcal/mol (Table 3). Then, the nonelectrostatic contribution was calculated from eq 3 as -8.11 kcal/mol using the overall binding free energies (∆G) of ligand-DNA interactions, which were obtained from the ITC experiments (Table 3), which revealed that the nonelectrostatic effect contributed greatly to lumiflavin-DNA binding. 3.4. Isothermal Titration Calorimetric (ITC) Studies. A complete thermodynamic profile of the interaction of small ligands with macromolecules is usually obtained by isothermal titration calorimetry.52-55 The binding constants (K), the binding stoichiometry (N), the enthalpy change (∆H), the entropy change (∆S), and the free-energy change (∆G ) ∆H - T∆S) can all be obtained using a single ITC experiment. To facilitate an unambiguous understanding of the nature of binding interactions of flavins with AP-site-containing DNA, we carried out ITC experiments on our ligand-DNA systems. A known volume of AP-site-containing DNA duplex having target thymine (5′-

Figure 4. Effect of salt concentration on the binding constant of lumiflavin (9) with an AP-site-containing DNA duplex having target thymine base. For comparison, data for riboflavin (b) are also shown. The DNA duplex used was 5′-d(TCC AGX GCA AC)-3′/(3′-d(AGG TCT CGT TG)-5′; X is Spacer C3. [Sodium cacodylate buffer] ) 10 mM; [EDTA] ) 1 mM; λex ) 448 nm for riboflavin and 443 nm for lumiflavin; temperature ) 278 K; pH ) 7.0; [lumiflavin] ) [riboflavin] ) 1 µM; [DNA] ) 0.2-5.0 µM.

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TABLE 3: Thermodynamic Parameters for the Binding of Flavins with an AP-Site-Containing DNA Duplex Having Target Thymine Base As Obtained from ITC Experiments at 278 K and the Corresponding Electrostatic and Nonelectrostatic Contributions Towards Their Total Binding Free Energy ligand

N

riboflavin lumiflavin

1.05 1.28

a

K (106M-1) 0.88 (0.054 5.5 (0.37

∆H (kcal/mol)

∆S (cal K-1/mol)

∆G(kcal/mol)

∆Gpea (kcal/mol)

∆Gtb (kcal/mol)

-11.54 -12.39

-14.3 -13.7

-7.56 -8.58

-0.56 -0.47

-7.0 -8.11

Using eq 2. b Using eq 3.

the AP site, the fact that lumiflavin binds strongly with the DNA over riboflavin is hence clearly evident. Apparently, among the four kinds of duplexes, the thermal stability of the DNA containing target thymine is effectively increased in the presence of either of the ligands, indicating the incorporation of these ligands into the AP site. Also, on the basis of ∆Tm values, we can see that lumiflavin binds more strongly to the DNA compared to riboflavin. In accordance with the results of Tm measurements (Table 1), the 1:1 binding constant (K11) values (Table 2) calculated from fluorescence titration experiments are higher when lumiflavin binds to the AP-site-containing DNA with thymine target. At 20 °C, in solutions buffered to pH 7.0 (I ) 0.11 M), K11 reaches 1.2 × 106 M-1 for lumiflavin-DNA complexation. This value is remarkably higher than the K11 of 2 × 105 M-1 reported earlier for riboflavin-DNA complexation.35 K11 for the binding of lumiflavin with DNA having cytosine target is calculated to be 3.9 × 105 M-1, implying a weaker binding of lumiflavin with DNA having target cytosine. Interestingly, the above value is noticeably higher than the 1:1 binding constant reported for the binding of riboflavin with DNA of identical sequence (8.1 × 104 M-1).35 With adenine or guanine targets, we observe negligibly weak binding for lumiflavin, though the K11 values were higher than those reported previously for the complexation of riboflavin with identical DNA sequences.35 Hence, in agreement with Tm measurements, lumiflavin exhibits stronger efficiency over riboflavin toward binding with DNA of identical sequence. 4.2. Nature of Nucleobase Recognition by Flavins. It is noteworthy that flavins, with hydrogen-bonding groups perfectly suited for hydrogen bonding with adenine, show less affinity to adenine in our system and recognize thymine. Recognition of flavins by adenine has been utilized by many RNA aptamers targeted toward flavins.56,57 In such cases, an adenine coplanar with flavins recognizes the polar groups along the edge of the isoalloxazine ring through hydrogen bonding wherein the uracillike edge of isoalloxazine hydrogen bonds to adenine.57 However, we observe from Tm and fluorescence measurements that flavins recognize thymine opposite the AP site; negligible affinity is observed toward DNA with an adenine target. As discussed previously, in our ligand-based SNP typing, it is expected that small hydrogen-bonding ligands are incorporated into the AP sites by stacking of the ligands with nucleobases flanking the AP site and by hydrogen bonding with target nucleobases. The formation of hydrogen bonding between flavins and target thymine is confirmed by 1H NMR spectroscopy measurements (Figure 6). As shown in Figure 6, the signals for the imino protons due to G-C base pairing are observed at 12.86 and 12.57 ppm for the 11-meric DNA duplex (spectrum a), where riboflavin/lumiflavin alone does not generate any signals (not shown). Upon complexation with riboflavin, new signals appear at 12.18 and 11.75 ppm accompanied by upfield shifts of the signals of the imino protons due to G-C base pairing (spectrum b). For lumiflavin-DNA complexation, the new signals appear at 11.76 and 11.57 ppm (spectrum c). These new signals can be assigned to the imino protons due to the binding

of thymine with the ligands, as has been observed for mismatched base pairings such as TT, which gave signals in a more upfield region as compared to those from fully matched base pairs.42 It is therefore likely that at the AP site, a pseudobase pair is formed with thymine base along the uracil-like edge of the isoalloxazine ring of the flavins. We also looked into the possibility of tautomerization of the ligands, which can, in principle, take place and alter the hydrogen-bonding face. Although flavins are known to undergo oxidation/reduction depending on pH, tautomerization does not occur under the physiological conditions.58 In our experiments, we first analyzed the binding behavior of the ligands by UV-vis titration experiments. We also examined the behavior by means of excitation spectra. Absorption and excitation spectra match each other well, which can rule out any excited-state interaction. In order to further understand the nature of flavin binding at the AP sites, we consider the influence of nucleobases flanking the AP site on the fluorescence of AP-site-incorporated flavins. The effect of all 16 kinds of flanking sequences to the AP site on the fluorescence intensity of flavins was examined (Figure S2, Supporting Information), wherein the target nucleobase was thymine. It was found that the most effective quenching took place for -GXG- (X ) AP site), indicating that flanking nucleotides significantly control the signaling properties of these ligands at the AP site. Strong fluorescence quenching of riboflavin/lumiflavin with DNA is observed when guanine bases flanks the AP site. An appropriate environment for the binding of these ligands with target thymine base opposite the AP site is hence provided when two guanine bases flank the AP site. These results are indicative of a cooperative binding event, as was characteristic of some typical RNA aptamers,59 wherein strong binding of flavins at the AP site with target nucleobases is achieved not only by hydrogen-bond formation with target nucleobases but also by stacking interactions with the nucleobases flanking the AP site. The fluorescence quenching is likely to be due to the electron transfer between excited ligand and the neighboring nucleobases. As seen in Figure S2 (Supporting Information), fluorescence quenching of lumiflavin depends strongly on the nucleobases flanking the AP site. Fluorescence quenching is maximum when two guanine bases flank the AP site, which is consistent with an electron-transfer mechanism in which the lumiflavin serves as an electron acceptor and guanine, the most easily oxidized nucleobase, as the electron donor.60 In contrast, fluorescence quenching is considerably less when the flanking bases are thymine or adenine. We have recently found that stronger quenching occurs for two cytosine bases than for two guanine bases flanking the AP site and that fluorescence enhancement occurs for two adenine or thymine bases flanking the AP site when amiloride is used as a ligand.61 Accordingly, further studies are needed to clarify the detailed mechanism on fluorescence quenching at the AP site. Also, the possibility of conformational rearrangements in DNA upon binding of the ligand at the AP site cannot be ruled out. Any conformational reorientations of the DNA structure are expected to strongly alter its electronic behavior and can, in turn, affect its interaction with the binding ligand. Studies on the binding

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Figure 5. Binding isotherms for the calorimetric titration of riboflavin (a) and lumiflavin (b) with AP-site-containing DNA having target thymine base. The DNA sequence used was 5′-d(TCC AGX GCA AC)-3′/3′-d(AGG TCT CGT TG)-5′, where X ) Spacer C3. In (a), [riboflavin] ) 30 µM and [DNA duplex] ) 500 µM; in (b), [lumiflavin] ) 10 µM and [DNA duplex] ) 150 µM. [Sodium cacodylate buffer] ) 10 mM; [NaCl] ) 100 mM; [EDTA] ) 1 mM; pH ) 7.0; temperature ) 278 K.

events of lumiflavin were done by molecular modeling simulations. On the basis of the structures calculated by MacroModel ver 9.0 with Amber* force field (Figure S3, Supporting Information), the isoalloxazine ring is located at the AP site, where the ring moiety is stacked with two flanking guanine bases. In addition, a pseudobase pair is formed with target thymine and cytosine along with the uracil-like edge of the isoalloxazine ring, whereas coplanar binding is restricted for adenine and guanine opposite the AP site. The AP site can give confinement for a ligand to be situated at the AP site, and thus, riboflavin, which has a long substituent, might receive steric hindrance, compared to lumiflavin. 4.3. Energetics of Flavin-DNA Interactions. The 1:1 binding of flavins to DNA is evident from ITC measurements (Table 3) and is also supported by the fluorescence titration experiments. The thermodynamic feasibility of complexation is apparent from the negative values of free energies (∆G) associated with the complex formation (-7.56 and -8.58 kcal mol-1, respectively, at 278 K for the complexation of riboflavin and lumiflavin with DNA). As seen in Table 3, the enthalpy change (∆H) associated with riboflavin-DNA binding (target thymine) is -11.54 kcal mol-1, and that for lumiflavin-DNA complexation is -12.39 kcal mol-1 Under the same conditions, the entropy changes (∆S) associated with the complexation events are -14.3 cal K-1 mol-1 for riboflavin-DNA complexation and -13.7 cal K-1 mol-1 for lumiflavin-DNA complexation. These thermodynamic parameters unambiguously indicate that the binding interactions of flavins at the AP site are enthalpy-driven, which suggests the stacking interaction of flavins at the AP site with flanking nucleobases. We calculated electrostatic contributions toward the total binding free energy of flavins with DNA from the ionic strength dependency of flavin binding with DNA (Table 3). The ionic strength-dependent fluorescence experiments at 278 K show that

Figure 6. 1H NMR spectra (in 95:5 H2O/D2O) showing the imino proton region of the AP-site-containing 11-mer DNA duplex (5′-d(TCC AGX GCA AC)-3′/3′-d(AGG TCT CGT TG)-5′; X ) Spacer C3) with target thymine base in the absence (a) and in the presence of riboflavin (b) and lumiflavin (c) supporting the formation of hydrogen bonds between the target nucleobase and the flavins. [DNA duplex] ) 0.80 mM; [riboflavin] ) 0.82 mM; [lumiflavin] ) 0.87 mM; [Tris buffer] ) 10 mM; [NaCl] ) 100 mM; [EDTA] ) 1 mM; pH ) 8.3; temperature ) 288 K.

1528 J. Phys. Chem. B, Vol. 113, No. 5, 2009 the apparent charge on lumiflavin when bound to DNA with target thymine is 0.45. The polyelectrolyte contribution (∆Gpe) toward the overall binding free energy for lumiflavin-DNA complexation is hence calculated as -0.47 kcal mol-1. Under the same conditions, the apparent charge on riboflavin when bound to an identical DNA sequence was 0.52 with a polyelectrolyte contribution of -0.56 kcal mol-1 toward the total binding free energy.35 These values reveal an effective contribution of ∆Gpe to the overall binding free energy of flavins when binding to DNA. Using eq 3, we calculated the nonelectrostatic contributions (∆Gt) to the overall binding free energies of flavin-DNA binding and obtained -7.0 and -8.11 kcal mol-1, respectively, for the complexation of riboflavin and lumiflavin with DNA (Table 3). These values reveal that nonelectrostatic effects contribute greatly toward the binding of flavins at the AP sites and are fundamental to the stabilization of flavin-DNA complexes. The observed free-energy changes for ligand-DNA interactions are generally assumed to be the sum of at least five freeenergy terms, as given by the following relationship62-65

∆Gobs ) ∆Gconf + ∆Gr+t + ∆Ghyd + ∆Gpe + ∆Gmol (4) where ∆Gconf is the contribution due to conformational transitions within the DNA and the ligand, ∆Gr+t is the contribution from the loss of rotational and translational freedom associated with bimolecular associations, ∆Ghyd represents the free energy of hydrophobic transfer of the ligand into the DNA binding site, ∆Gpe is the polyelectrolyte contribution, and ∆Gmol is the contribution from intermolecular DNA-ligand interactions within the binding site. For ligand-DNA complex formation, ∆Gr+t ) T∆Sr+t, where ∆Sr+t ) 50((10) cal mol-1 K-1, is generally agreed upon.65 Hence, ∆Gr+t for flavin-DNA binding is 13.9 kcal mol-1 at 278 K. The free energy due to hydrophobic interactions can be calculated using the expression ∆Ghyd ) 80((10)∆Cp,59 where ∆Cp is the experimentally determined heat capacity change. From a linear plot of ∆H, obtained from ITC measurements at various temperatures, versus the measurement temperatures, we determined ∆Cp values for flavin binding at the AP sites of DNA duplexes. The experimentally determined ∆Cp values are -197.6 and -198.8 cal mol-1 K-1, respectively, for the complexation of lumiflavin and riboflavin with DNA. These values are close to that for DNA intercalators.9 Applying these values, we calculated the hydrophobic contributions (∆Ghyd) toward the total binding free energy of flavin-DNA binding and obtained -15.81 and -15.9 kcal mol-1, respectively, for the complexation of lumiflavin and riboflavin with DNA. Substituting all known individual free-energy contributions into eq 4, the contributions together from the conformational transitions and molecular interactions (∆Gconf + ∆Gmol) for flavin-DNA binding can be calculated. The calculated values are -6.2 and -5.0 kcal mol-1, respectively, for lumiflavin and riboflavin complexation with DNA. The favorable hydrogen-bonding and stacking interactions between flavins and AP-site-containing DNAs are included in ∆Gmol. The thermodynamic profile constructed using the individual free-energy contributions for flavin-DNA binding is shown in Figure 7. It is now clear that the binding of flavins at the AP site is favorably driven by polyelectrolyte, nonelectrostatic, hydrophobic, and combined conformational and molecular effects, though unfavorable contributions exist from the reduction of rotational and translational freedom associated with the bimolecular complexation. The transfer of hydrophobic flavins from

Sankaran et al.

Figure 7. Thermodynamic profile for the incorporation of flavins into AP sites of DNA.

aqueous solution into the hydrophobic AP site is expected to be energetically favorable. This, along with the polyelectrolyte and nonelectrostatic free-energy contributions, determines the feasibility of flavin-DNA complexation. It may be presumed that the presence of water molecules inside of the AP site has a significant influence on the contributions due to molecular interactions. The formation of hydrogen bonds between the ligands and the nucleobase opposite the AP site may first involve breaking of hydrogen-bonded water molecules at the AP site. The competitive nature of these interactions, perhaps, is easily overtaken by the hydrophobic nature of the ligands and the hydrophobic nature of the AP site pocket. Further studies are necessary for a complete evaluation of various factors that facilitate a strong binding of flavins at the AP sites in DNA duplexes. 5. Conclusions The binding efficiency of lumiflavin incorporated into the AP sites of DNA duplexes with various target nucleobases was found to be significantly stronger than that previously reported for riboflavin, qualifying it as a potential candidate for efficient detection of thymine-related single-base mutations. Flavins bound to target nucleobases in DNA by hydrogen bonding. The incorporation of flavins into the AP site was facilitated by stacking interactions with nucleobases flanking the AP site, wherein two guanine bases that flanked the AP site provided the appropriate microenvironment for efficient binding. The binding of flavins with target nucleobases was largely favored by hydrophobic free-energy contributions together with nonelectrostatic and effective polyelectrolyte effects. Our efforts continue to clarify and to make use of such binding events, with a goal of developing flavins as potential candidates for SNP typing. Acknowledgment. This work was partially supported by Grants-in-Aid for Scientific Research (A) (No. 17205009) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Dr. Takeyoshi Kondo (Instrumental Analysis Center for Chemistry, Graduate School of Science, Tohoku University) for performing 1H NMR spectroscopic measurements. Supporting Information Available: Fluorescence titration curve for lumiflavin, the flanking base effect, and molecular

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