12628
J. Phys. Chem. B 2001, 105, 12628-12633
Competitive Binding Studies of H2T4 with DNA Hairpins (H2T4 ) meso-Tetrakis(4-(N-methylpyridiniumyl))porphyrin) Keith E. Thomas and David R. McMillin* Department of Chemistry, 1393 Brown Building, Purdue UniVersity, West Lafayette, Indiana 47907-1393 ReceiVed: May 15, 2001; In Final Form: September 27, 2001
Cationic porphyrins such as meso-tetrakis(4-(N-methylpyridiumyl))porphyrin, or H2T4, are remarkably versatile DNA binding agents of interest for potential therapeutic applications. This study focuses on the influence the DNA bases have in determining the mode of uptake and the relative binding affinity. For convenience the host is usually a DNA hairpin formed spontaneously by a 16-mer with the sequence 5′-GAXYACTTTTGTY′X′TC-3′, where X and Y designate varying nucleotides with complements X′ and Y′. Results from absorbance, emission, and CD spectroscopies lead to several conclusions. Among these is the recognition that the base composition, far more than the base sequence, dictates the mode of binding. Thus, any run of DNA containing at least 50% GtC base pairs supports intercalative binding simply because a robust hydrogenbonding framework (1) stabilizes the intercalated adduct, and (2) inhibits distortions that favor external binding. Another striking finding is that there is hardly any difference in binding constant for a hairpin that supports intercalation as compared with one that supports external binding, despite the host of factors that go into the energy balance. Finally, in and of itself, the steric bulk of the thymine methyl group does not prevent H2T4 from intercalating in AdT-rich regions of DNA because uridine-for-thymine base replacement has no effect on adduct formation.
Introduction
SCHEME 1
There is great interest in the cationic porphyrin (H2T4 ) meso-tetrakis(4-(N-methylpyridiniumyl))porphyrin, Scheme 1) because it is a novel DNA binding agent that could be a platform for applications in photodynamic therapy1-3 and/or virus control.4,5 This study focuses on the influence the DNA bases have in determining the mode of uptake and the relative binding affinity. Although certain conditions permit some type of hostinduced aggregation,6,7 the H2T4 ligand generally binds to DNA as a monomer.8-11 Hydrophobic effects may influence the binding affinity, but the work of Sari and co-workers has clearly established that Coulombic forces play an extremely important role.12,13 The H2T4 system is versatile in that it can either bind externally, in one and/or the other groove of B-form DNA, or intercalate between base pairs. The binding is very specific in that H2T4 only intercalates into guanine-cytosine (GtC) rich regions of DNA, whereas binding is external within domains that are rich in adenine-thymine (AdT) base pairs.14-16 NMR data presented by Marzilli and co-workers suggested that the specificity was even higher and that H2T4 intercalated in a highly sequence dependent fashion as well.17 In fact, they originally concluded that a 5′-CpG-3′ step was the only site that supported intercalative binding of the porphyrin. However, subsequent studies have indicated that other steps also allow intercalation of H2T4 or metal-containing derivatives, such as Cu(T4), which do not require axial ligands.18-21 The binding specificity is nevertheless quite impressive and may be largely a consequence of steric effects. One of the earliest realizations was that passage between base pairs would be difficult because the pyridinium substituents tend to be out of the plane of the porphyrin core.1 Even so, intercalation has proven to be a feasible binding motif, although the bulkiness of the porphyrin * Corresponding author fax: 765-494-0239; e-mail
[email protected] has a definite impact on the rates of insertion and deinsertion.14 Later, molecular dynamics calculations by Ford et al. indicated that contacts in the major groove involving thymine methyl groups and pyridinium groups of H2T4 could inhibit full intercalation of the porphyrin into a 5′-TpA-3′ step.22 Relatively recently, Williams and co-workers identified perhaps the most critical steric problem that occurs with intercalation of the porphyrin.23 By solving the crystal structure of a bound form of Cu(T4), they established that intercalative binding produces significant steric clashes in the minor groove involving pyridinium groups of the porphyrin and the sugar-phosphate chains of the DNA. In principle, binding constants for H2T4 interacting with various forms of DNA can provide important information about base and sequence preferences. However, the association constants are quite high, (ca. 106-107 M-1 with the DNA concentration expressed in terms of base pairs), hard to measure
10.1021/jp011860k CCC: $20.00 © 2001 American Chemical Society Published on Web 11/22/2001
Competitive Binding Studies of H2T4 with DNA accurately, and difficult to compare quantitatively.12,24 Thus, some workers have found comparatively high binding constants for GtC rich forms of DNA,25 while others have concluded that external binding to AdT rich sequences is equally favorable.14,24 Cooperative binding interactions also confuse the picture at high loadings, i.e., [H2T4]/[DNA] concentration ratios.14,19 The dearth of suitable DNA hosts is also a limitation. Thus, working with short, well-defined oligos is problematic because of competing association with single-stranded forms.26 Consequently, most studies have focused on variable-length, synthetic polymers, especially [poly(dG-dC)]2 and [poly(dAdT)]2, or random sequence material derived from calf thymus DNA. To avoid some of these problems, the studies described below focus on relative binding constants for H2T4 under lowloading conditions, where only 1:1 adducts are present. As was the case with Cu(T4),27-29 DNA hairpins prove to be convenient substrates. The results confirm that the binding constants are very similar for intercalation or external binding of H2T4, and they prove that the base composition is much more important than the base sequence in controlling the mode of binding. Experimental Section Materials. Integrated DNA Technologies (IDT, Coralville, IA) supplied all oligonucleotides, complete with a 5′-dimethyltrityl protecting group. The Poly-Pak purification cartridges were from Glen Research (Sterling, VA). Worthington Biochemical Corporation (Lakewood, NJ) supplied DNase I, while the H2T4 porphyrin came from Midcentury Chemical Company (Posen, IL). The dichlorodimethylsilane was a product of Sigma Chemical Company (St. Louis, MO). All other chemicals were reagent grade products from standard suppliers. Methods. Elution from the Poly-Pak cartridges according to manufacturer instructions yielded oligonucleotides suitable for quantitative work. A published procedure involving the digestion of the oligonucleotides with DNase I in a pH 7.9 Tris chloride buffer at 37 °C provided estimates of the molar extinction coefficients of the various hairpins at 260 nm (260).30 For all competition studies, the buffer was a µ ) 0.1 M, pH 6.8 phosphate buffer containing 0.05 M KCl. A siliconization pretreatment helped minimize adsorption of H2T4 to glass surfaces. The slit settings were 10 nm for all emission and emission lifetime experiments. Incorporation of a 510 nm long wave-pass filter blocked scattered light during the steady-state emission measurements. For ease of comparing relative emission intensities, the excitation was around 430 nm at a point where the absorbance of the free porphyrin matched that of the hairpin adduct. Instrumentation. The absorption and emission data came from Cary 100 Bio and SLM Aminco SPF500 C spectrophotometers, respectively. A JASCO J- 810 spectropolarimeter yielded the CD data at room temperature. A flashlamp-based Photon Technologies Inc. Model LS-100 system provided fluorescence lifetimes, and the pH meter was a Radiometer model PHM 64. Results Spectral Characterization. Interaction with a DNA hairpin induces a red shift in the Soret band of H2T4. Table 1 includes the stem sequence and the shorthand name for each hairpin substrate. For a micromolar total porphyrin concentration, greater than 98% of the porphyrin binds to DNA if the binding constant is of the order of 106 M-1 and the base-pairs-toporphyrin ratio is at least 40:1. Consistent with this estimate, the absorbance data in Figure 1 show that a 5:1 hairpin-to-
J. Phys. Chem. B, Vol. 105, No. 50, 2001 12629 TABLE 1: Hairpin Substrates and Physical Data for H2T4 Adducts in µ ) 0.1 pH 6.8 Phosphate Buffer at Hairpin/ Porphyrin Ratios of 5:1 CD extremum hairpin stema
name
5′-GATTAC {TT} 3′-CTAATG 5′-GATAAC {TA} 3′-CTATTG 5′-GAUAAC {UA} 3′-CUAUUG 5′-GACGAC 3′-CTGCTG 5′-GAGCAC 3′-CTCGTG 5′-GAAGAC 3′-CTTCTG 5′-GAAGAC 3′-CTTCUG 5′-GAAGAC 3′-CTUCTG
∆λc λ ∆ 260b mM-1 cm-1 nm %Hd nm M-1 cm-1
{CG}
Externally Bound H2T4 142 8 10
425
40
150
8
3
425
50
143
8
3
425
42
Intercalated H2T4 135 18
38
439
-22
{GC}
132
18
38
439
-12
{AG}
138
15
29
436
-12
12-U-{AG}
138
15
29
438
-9
14-U-{AG}
138
15
29
436
-9
a In each case the loop connecting residues C6 and G11 is TTTT. Extinction coefficient of free hairpin at 260 nm. c Red shift of the Soret maximum from 424 nm for free H2T4. d Percent change of absorbance of the Soret maximum.
b
Figure 1. Soret band of H2T4 in pH 6.8 phosphate buffer. The {TT}/ porphyrin ratios are 0 (A), 5 (B), 10 (C), and 15 (D).
porphyrin concentration ratio is sufficient to drive adduct formation to completion in the case of the TT hairpin. The same is true with the other hairpins, despite the fact that two different types of adducts occur. Thus, the data in Table 1 indicate that adducts with AdT rich hairpins such as {TT} and {TA} characteristically exhibit modest shifts of the absorption maximum in the Soret region (∆λ ≈ 8 nm) along with weak hypochromism (H e 10%) and induction of a positive CD signal. In contrast, adduct formation with a GtC-rich hairpin such as {CG} or {GC} induces a CD signal with the opposite sign, a much larger spectral shift (∆λ g 15 nm), and more pronounced hypochromism (H g 30%). Interaction with a single-stranded 8-mer of adenine nucleotides produces changes in absorption similar to those observed with {GC} or {CG}. The spectral shift is somewhat smaller, but the induced hypochromism is quite significant (Figure 2). The CD results also confirm that formation of the 1:1 adduct is complete at a 5:1 hairpin/porphyrin ratio. In addition, there is evidence for a competing type of adduct formation with AdT rich hairpins when the porphyrin is in excess. Thus, Figure 3 shows that a solution of the {TT} hairpin gives a conservative
12630 J. Phys. Chem. B, Vol. 105, No. 50, 2001
Figure 2. Soret band of H2T4 perturbed by the addition of the singlestranded oligonucleotide octa-adenine in pH 6.8 phosphate buffer with oligomer/porphyrin ratios of 0 (A), 5 (B, thick), 20 (C), and 75 (D, thick).
Figure 3. CD signal from H2T4 bound to the {TT} hairpin in pH 6.8 phosphate buffer with hairpin/porphyrin ratios of 0.4 (A), 0.8 (B, thick), 2 (C), and 5 (D, thick).
Thomas and McMillin
Figure 5. Emission spectra of H2T4 in pH 6.8 phosphate buffer. A, thick: in the absence of DNA; B, thick: in the presence of {GC}; C: in the presence of {AG}; and D: in the presence of {TT}. The hairpin/ porphyrin ratios are 5:1.
Figure 6. Soret absorption of H2T4 in pH 6.8 phosphate buffer containing A:{TT}; B: {CG}; or C, thick: {TT} and {CG}. For each type of DNA present the hairpin/porphyrin ratio is 5:1.
TABLE 2: Physical Data for H2T4 Bound to DNA Hairpins in µ ) 0.1 pH 6.8 Phosphate Buffer hairpin
Krel, M-1
λaem, nm
φrelb
τc, ns
8.6
{TA} {TT}
Externally Bound H2T4 1.1 653, 715 0.8 653, 715
3.0 3.6
{CG} {GC} {AG} 12-U-{AG} 14-U-{AG}
Intercalated H2T4 1.0 662, 725 1.0 660, 724 0.5 660, 723 0.5 660, 723 0.5 601, 723
1.0 1.0 1.3 1.1 1.5
4.2 5.8
a Emission maxima corrected as before: Eggleston, M. K.; McMillin, D. R.; Koenig, K. S.; Pallenberg, A. J. Inorg. Chem. 1997, 36, 172176. b Relative emission quantum yield. c Fluorescence lifetime.
Figure 4. Overlay of CD signals from H2T4 bound to the A: {TA}; B, thick: {UA}; C, {GC}; D, thick: {AG}; and E: {CG} hairpins in pH 6.8 phosphate buffer at a 5:1 hairpin/porphyrin ratio.
CD signal at a hairpin/porphyrin ratio of 0.4 or less, with a crossover occurring at the same wavelength as the absorption maximum. Upon addition of more hairpin, the signal evolves into a positive band with a maximum at a slightly shorter wavelength than the absorption maximum. The same is true of the other AdT rich hairpins. In contrast, the absorption and CD signals occur at the same wavelength when adduct formation is with a GtC rich hairpin. See Figure 4 for an overlay of representative CD spectra and Table 1 for a compilation of results.
The emission signal from H2T4 also depends on the hairpin host, but the variations are less dramatic. The emission sharpens upon adduct formation, independent of the composition of the host (Figure 5). However, the emission from an adduct involving an AdT rich hairpin occurs at somewhat shorter wavelengths, is more intense, and exhibits a longer lifetime (Table 2). Competition Studies. The spectral properties of a solution containing H2T4 and two different hairpins is a simple reflection of the two types of adducts in equilibrium. Figure 6C, a typical example of absorption by a mixture, contains H2T4, {TT}, and {CG}. Spectra A and B in Figure 6 are the control spectra for the individual adducts. Corresponding CD data appear in Figure 7. If letters A and B are labels for the two hairpins in solution,
Competitive Binding Studies of H2T4 with DNA
J. Phys. Chem. B, Vol. 105, No. 50, 2001 12631
Figure 7. CD signal from H2T4 in pH 6.8 phosphate buffer containing A: {TT}; B: {CG}; or C, thick: {TT} and {CG}. As before, the hairpin/porphyrin ratio is 5:1 for each type of DNA.
one can easily resolve the spectrum of the mixture, i.e., spectrum C, into a weighted sum of the spectra of the contributing adducts. By design, the DNA concentration is high enough to sequester all of the porphyrin, and the total porphyrin concentration is the same in all solutions. Therefore, if WA represents the fraction of porphyrin bound to hairpin A, and 1 - WA designates the fraction bound to hairpin B, the ratio R of the concentration of the adduct with hairpin A over that of the adduct with hairpin B is
R)
WA 1 - WA
(1)
Furthermore, if KA and KB designate the respective equilibrium constants for formation of adducts with hairpins A and B,
[
CB(1 + R) - PT KA )R KB CA(1 + R) - R‚PT
]
(2)
where CA, CB, and PT denote, respectively, the total concentrations of hairpin A, hairpin B, and porphyrin. For a series of hairpins of interest, Table 2 contains relative equilibrium constants obtained via eq 2 with {CG} as the common reference. The estimated precision of the KA/KB ratio is ( 0.1. Discussion Adduct Formation. Since the loops are so tightly structured in DNA hairpins of the type studied here,31,32 one would expect H2T4 to bind selectively in the stem regions as previously established for the analogous copper derivative Cu(T4).29 In line with expectations, the data in Table 1 indicate that H2T4 forms the same two types of adducts as Cu(T4). Thus, with AdTrich hairpins adduct formation induces a positive CD signal and relatively minor changes in the Soret absorption band, consistent with external, or groove, binding.8,10,11 On the other hand, interaction with a GtC-rich hairpin induces a negative CD signal as well as a pronounced red shift and strong hypochromism in the Soret region, all clear signs of intercalation.8,10,11 Intercalative binding does not require a 5′-CpG-3′ step or even the presence of contiguous GtC base pairs because H2T4 readily intercalates into {GC} as well as {AG}. As with Cu(T4),27 a local composition of 50% GtC base pairs is sufficient to support intercalative binding of the porphyrin. It is important to note that the data do not define precisely where intercalation occurs within the stem. However, the great advantage of intercalative binding is that it allows the duplex
structure to remain intact; hence, it is logical to assume that the porphyrin inserts next to strongly hydrogen-bonded GtC base pairs as much as possible in order to minimize any straininduced distortions. In line with this reasoning, a comparison of the Soret data for the {AG}, {CG}, and {GC} adducts reveals that both the ∆λ and %H of adduct formation are greater for hairpins with contiguous GtC base pairs in their stems. Relative Binding Constants. The relative binding constants in Table 2 provide useful comparisons of the host-guest interactions. One intriguing finding is that H2T4 exhibits practically the same affinity for hairpins with AdT-rich or GtC-rich DNA sequences.14,24 The data also confirm the preeminent role the base composition has in directing the binding. Thus, H2T4 intercalates with equal affinity into a sequence containing a 5′-GpC-3′ step or a 5′-CpG-3′ step. The binding constant for {AG} is significantly lower, yet the spectral results show that intercalation is still the preferred mode of binding. To understand the latter result, one must bear in mind all of the forces, repulsive and resistive alike. For convenience one can analyze the energetics in terms of the following equation:
∆G ) ∆GR + ∆GB
(3)
where ∆G represents the interaction energy between the porphyrin and the hairpin, ∆GR is the energy required to reorganize the DNA into the final binding configuration, and the ∆GB term includes all other energy effects including the (noncovalent) bond energy, solvation changes, etc. Typically, the ∆GR term is modest for simple intercalative binding as the reorganization involves only a natural unwinding motion of the DNA double helix.33 Accordingly, heteroaromatic cations ordinarily bind by intercalation as long as the ∆GB term, which includes hydrophobic effects, is reasonably favorable. For a bulky porphyrin such as H2T4, however, steric interactions within the minor groove reduce ∆GB, so much so that external binding becomes the favored motif amidst flexibile DNA sequences. To the degree that partial melting of the DNA structure provides for a molded, or “induced”, fit of the bulky porphyrin,19 the ∆GB term may actually favor external binding of H2T4, regardless of the base composition. The ∆GR term is therefore decisive. It is evidently too positive for external binding to hosts such as {AG}, which contain at least 50% GtC base pairs.28 Specific interactions may also play a role, if intercalation next to guanine-cytosine base pairs enhances the ∆GB term. Other intercalators exhibit that same base preference.34-36 Such an effect could help explain the relative affinities for {CG} or {GC} versus {AG} since more guanine/porphyrin stacking interactions are possible with {CG} or {GC}. Specific interactions can also be destabilizing, e.g., a steric clash with the thymine methyl substituent when H2T4 intercalates next to adenine/thymine base pairs, but see below. Uridine Replacement. According to the molecular dynamics calculations of Ford et al., a 5′-ApT-3′ step is incapable of internalizing H2T4 in the same way as a 5′-CpG-3′ step due to steric congestion in the major groove.22 More specifically, their calculations suggest that complete insertion of the porphyin would lead to clashes involving the C4 methyl substituents of the thymines and the pyridinium side chains of the guest. However, steric interactions with the thymine methyls are not the primary reason for the absence of intercalative binding in AdT-rich sequences because spectral data in Table 1 reveal that H2T4 still binds externally when uridine is present in place of thymine. A further test of the steric influence of the thymine methyl revolves around {AG}, a hairpin that does support the
12632 J. Phys. Chem. B, Vol. 105, No. 50, 2001 intercalation of H2T4. The idea is to compare the binding affinities of {AG} and the two modified forms shown below.
If, as expected, the porphyrin intercalates next to the central guanine/cytosine base pair, either 14-U-{AG} or 12-U-{AG} should show enhanced affinity if interaction with the thymine methyl substituent is an important factor. However, there is no measurable difference in affinity for any of the three hairpins (Table 2). Effects on Emission. As is evident in Figure 5, binding interactions with DNA hairpins affect the band shape as well as the energy of the emission from H2T4. The most obvious spectral change is the enhanced vibrational structure. Vergeldt and co-workers have suggested that the (solvent-dependent) broadening of the emission from the free porphyrin ultimately traces back to the distribution of torsion angles available to the pyridinium substituents.37 The situation is likely to be very different in the DNA adducts for two reasons. One is that binding forces will impose a more rigid structure on the porphyrin and thereby constrain the torsional motion of the substituents. In addition, the local “solvent” environment will be very different for the bound porphyrin. Although the band shapes are similar for both bound forms, the emission maxima of the intercalated porphyrin occur at longer wavelengths. The absorption spectra show similar trends, but the differential shift is much larger in the Soret region, in either energy or wavelength terms. Barnes et al. previously noted that the absorbance shift is smaller in the longer wavelength Q-band region,38 and they attributed the effect to differences in orbital participation. However, in the first approximation, the Soret and Q-band excitations represent different admixtures of the same two electronic configurations.39 The absorption energy is probably an important consideration. In fact, to the extent that the shifts arise from exciton-like coupling with excitations of the DNA bases, the bathochromic shift should weaken as the frequency gap between the oscillators widens. The emission intensity also varies significantly with the mode of binding and, therefore, the base content of the DNA. Kelly and co-workers previously studied the emission of bound H2T4 and suggested that the intercalated form was subject to reductive electron-transfer quenching by guanine residues.40 Consistent with this prediction, Jasuga et al. later showed that guanine moiety of the free nucleotide dGMP is an excellent quencher.41 However, the results in Figure 5 reveal that the presence of guanine only induces partial quenching in an intercalating host such as {AG} or {CG}. The results suggest that reductive quenching by guanine is an endergonic process within the hairpin stems; otherwise, quenching would be very efficient in short DNA hosts of this type.42-44 More favorable energetics for quenching by dGMP could be due to a difference in the geometry of the encounter complex and/or in local solvation. When net electron transfer is endergonic, quenching can also occur via partial electron transfer and excited-state complex (exciplex) formation, although there is no sign of a red-shifted emission here. In that scenario, the quenching efficiency would logically increase in proportion to the strength of coupling which, in turn, would vary with the number of guanine residues contacting the porphyrin. Thus, exciplex-induced quenching can
Thomas and McMillin SCHEME 2
also account for the different emission lifetimes of the {AG} and {CG} adducts. The emission yields and lifetimes in Table 2 follow parallel trends, but the results with the {TT} adduct are not in perfect proportion with those obtained with the {CG} and {AG} adducts. All else the same, the quantum yield varies with the lifetime according to the following equation:
φ ) k rτ
(4)
where kr denotes the radiative rate constant, φ the emission yield, and τ the lifetime. By comparison with the {CG} analogue, eq 4 predicts that the {TT} adduct should have a relative quantum yield of 2.0; however, the experimental value is 3.6 (Table 2). Disproportionate yields can occur if there are two intercalation sites, for example, one that provides for essentially complete quenching and another that supports partial quenching. Under such conditions eq 4 does not apply. However, complete quenching of any component of the emission would almost certainly require exergonic electron transfer from guanine, and that would mean total quenching because every intercalation site in the {CG} stem involves contact with at least one guanine residue. A better way to rationalize the disproportionate quantum yields is to recognize that kr can vary from host to host. Indeed, one would expect the {TT} adduct to exhibit a larger radiative rate constant (and a higher quantum yield) because the electronic transition is more highly allowed due to relatively weak hypochromism. Other Adducts. As reported previously for Cu(T4),29 the CD results show that a 2:1 adduct forms when H2T4 binds externally and there is a substoichiometric amount of hairpin in solution. More specifically, Figure 3 shows that under these conditions the system exhibits a conservative CD spectrum, in line with the occurrence of porphyrin/porphyrin interactions involving chromophores bound to the same host.6 With the addition of more hairpin, however, the figure also reveals that the H2T4 disperses until all that remains is the 1:1 adduct with its positive CD signal. A more intriguing finding is that H2T4 also forms two types of adducts with single-stranded DNA. Some time ago, Pasternack and co-workers observed that single-stranded poly (dA) takes up H2T4.45 In light of the hypochromic response and the bathochromic shift of the Soret band, they attributed the binding to pseudointercalation, i.e., interdigitation between adjacent bases of the chain. Lugo-Ponce and McMillin subsequently reported that Cu(T4) forms the same type of adduct,29 and the absorbance data in Figure 2 demonstrate that the 8-mer of adenine nucleotides also supports the pseudointercalation of H2T4. However, above an oligo-to-porphyrin ratio of about
Competitive Binding Studies of H2T4 with DNA 20:1, the Soret band shifts to even longer wavelength and the hypochromism weakens, consistent with formation of a new adduct by complexation with a second oligonucleotide in solution. Since repulsive forces are likely to dominate when anionic, noncomplementary oligonucleotides of this type come together, the porphyrin may sandwich between skewed runs of oligonucleotide, as suggested in Scheme 2. Formation of a related bridged structure has previously been proposed in order to account for viscometric data from solutions containing a hydrophobic bis(phenanthroline)copper(I) complex and B-form DNA duplexes.46 Conclusions Hairpin structures are useful substrates for studying the DNA binding interactions of H2T4. In accordance with results obtained with DNA polymers, the present studies show that the porphyrin binds externally to hairpins with stems enriched in AdT base pairs but intercalates into hairpins that have GtC-rich stems. Interestingly, there is hardly any difference in binding constant for a hairpin that supports intercalation as compared with one that supports external binding. Binding studies with different hairpin stems reveal that the base composition is much more important than the base sequence in determining the mode of binding. Thus, any run of B-form DNA containing at least 50% GtC base pairs will support intercalation of H2T4, as found previously for Cu(T4). One reason is that a robust hydrogen bonding framework enables the double helical structure to withstand the strain associated with internalization of the bulky porphyrin, particularly the steric clashes known to occur in the minor groove. The other side of the coin is that the rigidity of a GtC-rich duplex inhibits the kind of structural reorganization that appears to be necessary for high-affinity external binding. Uridine-for-thymine base replacement does not affect the mode of binding, hence the steric bulk of the thymine methyl group is not the major deterrent to intercalative binding in AdT-rich sequences. Emission intensity and lifetime measurements reveal that GtC base pairs quench the emissive excited state from intercalated forms of H2T4, but probably not by a simple electron-transfer mechanism. Finally, a somewhat surprising finding is that formation of a 2:1 oligo-to-porphyrin adduct is possible with single-stranded DNA substrates. Acknowledgment. The National Science Foundation supported this work through grant number CHE 01-08902. References and Notes (1) Fiel, R. J.; Howard, J. C.; Mark, E. H.; Datta-Gupta, N. Nucleic Acids Res. 1979, 6, 3093-3118. (2) Milgrom, L.; MacRobert, S. Chem. Ber. 1998, 34(5), 45-50. (3) Ali, H.; van Lier, J. E. Chem. ReV. 1999, 99, 2379-2450. (4) Dixon, D. W.; Schinazi, R.; Marzilli, L. G. Ann. N. Y. Acad. Sci. 1990, 616, 511-513. (5) Kasturi, C.; Platz, M. S. Photochem. Photobiol. 1992, 56, 427429. (6) Carvlin, M. J.; Datta-Gupta, N.; Fiel, R. J. Biochem. Biophys. Res. Commun. 1982, 108, 66-73. (7) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393-5399. (8) Fiel, R. J. J. Biomol. Struct. Dyn. 1989, 6, 1259-1274. (9) Marzilli, L. G. New J. Chem. 1990, 14, 409-420. (10) Pasternack, R. F.; Gibbs, E. J. Met. Ions Biol. Syst. 1996, 33, 367397.
J. Phys. Chem. B, Vol. 105, No. 50, 2001 12633 (11) McMillin, D. R.; McNett, K. M. Chem. ReV. 1998, 98, 1201-1219. (12) Sari, M. A.; Battioni, J. P.; Mansuy, D.; Lepecq, J. B. Biochem. Biophys. Res. Commun. 1986, 141, 643-649. (13) Sari, M. A.; Battioni, J. P.; Dupre, D.; Mansuy, D.; Lepecq, J. B. Biochemistry 1990, 29, 4205-4215. (14) Pasternack, R. F.; Gibbs, E. J.; Villafranca, J. J. Biochemistry 1983, 22, 2406-2414. (15) Ward, B.; Skorobogaty, A.; Dabrowiak, J. C. Biochemistry 1986, 25, 7827-7833. (16) Strickland, J. A.; Marzilli, L. G.; Gay, K. M.; Wilson, W. D. Biochemistry 1988, 27, 8870-8878. (17) Marzilli, L. G.; Banville, D. L.; Zon, G.; Wilson, W. D. J. Am. Chem. Soc. 1986, 108, 4188-4192. (18) Strickland, J. A.; Banville, D. L.; Wilson, W. D.; Marzilli, L. G. Inorg. Chem. 1987, 26, 3398-3406. (19) Gibbs, E. J.; Maurer, M. C.; Zhang, J. H.; Reiff, W. M.; Hill, D. T.; Malcika-Blaszkiewicz, M.; McKinnie, R. E.; Liu, H. Q.; Pasternack, R. F. J. Inorg. Biochem. 1988, 32, 39-65. (20) Hudson, B. P.; Sou, J.; Berger, D. J. M., D. R. J. Am. Chem. Soc. 1992, 114, 8997-9002. (21) Kruk, N. N.; Shishporenok, S. I.; Korotky, A. A.; Galievsky, V. A.; Chirnovy, V. S.; Turpin, P. Y. J. Photochem. Photobiol. B. 1998, 45, 67-74. (22) Ford, K.; Fox, K. R.; Neidle, S.; Waring, M. J. Nucleic Acids Res. 1987, 15, 2221-2234. (23) Lipscomb, L. A.; Zhou, F. X.; Presnell, S. R.; Woo, R. J.; Peek, M. E.; Plaskon, R. R.; Williams, L. D. Biochemistry 1996, 35, 2818-2823. (24) Strickland, J. A.; Marzilli, L. G.; Wilson, W. D. Biopolymers 1990, 29, 1307-1323. (25) Feng, Y.; Pilbrow, J. R. Biophys. Chem. 1990, 36, 117-131. (26) Blom, N.; Odo, J.; Nakamoto, K.; Strommen, D. P. J. Phys. Chem. 1986, 90, 2847-2852. (27) Eggleston, M. K.; Crites, D. K.; McMillin, D. R. J. Phys. Chem. A 1998, 102, 5506-5511. (28) Tears, D. K. C.; McMillin, D. R. Chem. Commun. 1998, 25172518. (29) Lugo-Ponce, P.; McMillin, D. R. Coord. Chem. ReV. 2000, 208, 169-191. (30) Nadeau, J. G.; Gilham, P. T. Nucleic Acids Res. 1985, 13, 82598274. (31) vanDongen, M. J. P.; Mooren, M. M. W.; Willems, E. F. A.; vanderMarel, G. A.; vanBoom, J. H.; Wijmenga, S. S.; Hilbers, C. W. Nucleic Acids Res. 1997, 25, 1537-1547. (32) Blommers, M. J. J.; Walters, J.; Haasnoot, C. A. G.; Aelen, J. M. A.; Vandermarel, G. A.; Vanboom, J. H.; Hilbers, C. W. Biochemistry 1989, 28, 7491-7498. (33) Calladine, C. R.; Drew, H. R. Understanding DNA; Academic: New York, 1997. (34) Muller, W.; Crothers, D. M. Eur. J. Biochem. 1975, 54, 267-277. (35) Hobza, P.; Sponer, J.; Polasek, M. J. Am. Chem. Soc. 1995, 117, 792-798. (36) Sundquist, W. I.; Lippard, S. J. Coord. Chem. ReV. 1990, 100, 293322. (37) Vergeldt, F. J.; Koehorst, R. B. M.; Vanhoek, A.; Schaafsma, T. J. J. Phys. Chem. 1995, 99, 4397-4405. (38) Barnes, N. R.; Schreiner, A. F.; Dolan, M. A. J. Inorg. Biochem. 1998, 72, 1-12. (39) Gouterman, M. In The Porphyrins; Dolphin, D., Ed.; Academic: New York, 1978; Vol. iii, part A, pp 1-165. (40) Kelly, J. M.; Murphy, M. J.; McConnell, D. J.; Ohuigin, C. Nucleic Acids Res. 1985, 13, 167-184. (41) Jasuja, R.; Jameson, D. M.; Nishijo, C. K.; Larsen, R. W. J. Phys. Chem. B 1997, 101, 1444-1450. (42) Wan, C. Z.; Fiebig, T.; Schiemann, O.; Barton, J. K.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14052-14055. (43) Meggers, E.; Dussy, A.; Schafer, T.; Giese, B. Chem.-Eur. J. 2000, 6, 485-492. (44) Lewis, F. D.; RL, L.; Wasielewski, M. Acc. Chem. Res. 2001, 34, 159-170. (45) Pasternack, R. F.; Brigandi, R. A.; Abrams, M. J.; Williams, A. P.; Gibbs, E. J. Inorg. Chem. 1990, 29, 4483-4486. (46) Liu, F.; Meadows, K. A.; McMillin, D. R. J. Am. Chem. Soc. 1993, 115, 6699-6704.
