J. Phys. Chem. B 1999, 103, 9321-9327
9321
Photophysical Quenching Mediated by Guanine Groups in Pyrenyl-N-alkylbutanoamide End-Labeled Oligonucleotides Eran Zahavy and Marye Anne Fox*,† Department of Chemistry and Biochemistry, UniVersity of Texas, Austin, Texas 78712, and Department of Chemistry, North Carolina State UniVersity, Raleigh, North Carolina 27695 ReceiVed: April 27, 1999; In Final Form: August 2, 1999
Two series of pyrenyl-N-alkylbutanoamide end-labeled oligonucleotides have been prepared as models for photophysical quenching along DNA segments. Pyrenebutanoic acid (P) has been attached through 6-aminohexyl or 3-aminopropyl linkers to the 5′ edge of an oligonucleotide composed of 10 deoxyadenosines (A) and one deoxyguanosine (G) at a defined site along each strand, P-NH-(CH2)3 or 6-5′-AnGA10-n-3′ (n ) 2-10). The complementary strand is composed of deoxythymidosines (T) and one deoxycytidosine (C) at the corresponding positions required for matched base pairing, 5′-T10-nCTn-3′ (n ) 2-10). This configuration has allowed us to investigate deoxyguanosine-induced quenching of pyrenebutanoamide fluorescence (through guanine to pyrenebutanoamide electron transfer or nonradiative internal conversion) along oligonucleotides in either single- or double-stranded forms. The observed quenching rates in the (CH2)6-linked series do not depend monotonically on the distance separating the excited pyrenebutanoamide from the deoxyguanosine quencher because a less efficient competing quenching by deoxythymidosine on the complementary chain complicates the kinetic analysis. The observed quenching efficiency along a DNA segment is significantly affected by the conformation of the appended quencher.
Introduction The investigation of electron transfer (ET) along DNA segments has been very intensive in the past several years,1-7 both to understand the mechanism of electron exchange along a DNA scaffold and to utilize DNA matrixes for the development of biosensors8 and bioelectronic9 devices. Although electron transfer in proteins does occur over long segments of bases (>10 Å) with a distance dependence similar to that reported for hydrocarbon spacers (0.8 Å-1 < β < 1.4 Å-1),10 long-range ETs along DNA have much lower reported β values, an observation for which a coherent interpretation is still being debated.11-13 For example, Barton et al. have found that in helical DNA in which inorganic donor and acceptor pairs are intercalated in DNA major grooves, photoinduced ET takes place over very long distances (up to 40 Å) and with a very low distance dependence (β ∼ 0.2 Å-1).1c-1e,4b Similarly, in a series of flash-quench experiments, an excited chromophore intercalated in DNA induces oxidation of a deoxyguanine (in -GG- sites) located up to 40 Å from the site of absorption, effecting a strand cleavage after treatment with piperidine.1e,4a Parallel observations have also been reported by Schuster et al.,14 Saito et al.,15 and Giese et al.16 In systems in which organic donor/acceptor pairs were used, somewhat larger β values were found. For example, in stilbenemodified hairpin duplexes, Lewis et al. found β ) 0.64 Å-1 for photoinduced ET from deoxyguanine to an end-bound excited stilbene.7c Tanaka et al. studied ET between acridine bound to an oligonucleotide sequence containing one or more deoxyguanines at varying distances and found a distance dependence similar to that observed in protein matrixes (β ) * Corresponding author. † Current address: Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204.
1.42 Å-1).3 With organic donor/acceptor pairs that were noncovalently attached to duplex DNA, Harriman et al. found β ) 0.88 Å-1.2 Several organic excited states are quenched by guanine when either appended to the same DNA or attached to an individual guanine derivative.3,7c Although fluorescence quenching of pyrene can take place through vibronically coupled internal conversion17,18 or through energy transfer to a forbidden n, π* transition,19 reported redox potentials are consistent with a primary mechanism for the pyrene-guanine interaction that involves electron transfer from deoxyguanine to the excited pyrene moiety. Despite the difficulty in measuring individual nucleoside reduction potentials when present on an oligonucleotide chain, the oxidation potentials of the nucleotide bases themselves have been evaluated recently by Steenken et al.20: dG+•/dG ) 1.29 V, dA+•/dA ) 1.42 V, dT+•/dT ) 1.7O V, dC+•/dC ) 1.6O V at neutral pH). From those values, guanine is the only base that can exothermically reduce an excited pyrene (*P/P-• ) 1.32 V)21 with a total free energy ∆G ) -0.03 V. The efficiency of this ET has been demonstrated experimentally in the photoexcitation of a pyrene group directly attached to a substituted guanine22 and it is reasonable to expect comparable potentials when a substituted pyrenylalklylcarboxamide is bound to the same DNA segment at a distance from the substituted guanosine in question. In other studies, it has been shown that thymidine and cytosine can act as electron acceptors for excited pyrene. For example, Netzel et al.6b and Mann et al.23 describe attaching a pyrene group directly to a thymidine which was then introduced into an oligonucleotide, resulting in quenching of the pyrene emission. Similarly, Geacintov et al.22c have demonstrated oxidative quenching of pyrene in pyrene-thymidine or pyrenecytosine couples, but only in protic (aqueous) solution where a
10.1021/jp9913822 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999
9322 J. Phys. Chem. B, Vol. 103, No. 43, 1999
Zahavy and Fox
TABLE 1: Fluorescence Quantum Yields and Lifetimes of a Series of Pyrenyl-N-hexylcarboxamide-Modified Oligonucleotides compound
fluorescence fluorescence quantum yields lifetimes (φ)(0.02 (ns)a
pyrenebutanoic acid P-NH-(CH2)6-5′-A10-3′ P-NH-(CH2)6-5′-A10-3′-3′-T10-5′ P-NH-(CH2)6-5′-A10G-3′ P-NH-(CH2)6-5′-A10G-3′-3′-T10C-5′
0.44 0.44 0.43 0.40 0.34
P-NH-(CH2)6-5′-A10C-3′ P-NH-(CH2)6-5′-A10C-3′-3′-T10G-5′
0.42 0.35
P-NH-(CH2)6-5′-A9GA-3′ P-NH-(CH2)6-5′-A9GA-3′-3′-T9CT-5′
0.43 0.32
P-NH-(CH2)6-5′-A4GA6-3′ P-NH-(CH2)6-5′-A4GA6-3′-3′-T4CT6-5′
0.40 0.27
P-NH-(CH2)6-5′-A2GA8-3 P-NH-(CH2)6-5′-A2GA8-3′-3′-T2CT8-5′
0.36 0.17
P-NH-(CH2)6-I‚I′b
0.13
180((2) 180((2) 162((2) 144((2) 79((2), 89% 456((4), 11% 156((2) 76((2), 90% 443((4), 10% 118((2) 74((2), 90% 352((4), 10% 119((2) 64((2), 80% 221((4), 20% 90((2) 14((2), 80% 126((4), 20% 6.0((0.2)
(I ) 5′-AAGTAGCAGC-3′). The efficiency of fluoroscence quenching of the appended pyrene derivative was then monitored for each oligonucleotide, both as a single strand and when complexed with the complementary chain, to study the role of single and multiple deoxyguanosines in long-range ET along these rigid scaffolds.
a
The relative contribution of each component is indicated as a percentage of the biexponential fit. b I ) 5′-AAGTAGCAGC-3′, I′ ) 5′-GCTGCTACTT-3′.
coupled, fast secondary proton transfer can ensue. These observations are also consistent with the reported reduction potentials for deoxythymidosine and deoxycytosine in the presence of a proton source (dC/dC-H• ) -1.36 V; dT/dTH• ) -1.35V).22b Lewis and co-workers7a,b have shown that pyrene dimer emission is also quenched by deoxythymidine, but with decreased efficiency. In the last several years, our group has studied photoinduced ET in several new macromolecules in order to define the photophysical principles that control vectorial energy and electron migration over distances that are large on a molecular scale. We have prepared several families of rigid linear polymers including synthetic polypeptides24a,25 that can accommodate pendent chromophores (such as substituted pyrenylalkylcarboxamides) at specific sites.24 Here we extend these studies to include photophysical quenching of pyrene end-labeled oligonucleotides by a specific deoxyguanosine positioned along a single oligonucleotide strand and in a paired oligonucleotide duplex. Results and Discussion Two series of pyrene-N-alkylbutanoamide-appended oligonucleotides have been prepared. In the first family, pyrenebutanoic acid 1 is attached, eq 1, through a 6-aminohexyl linker to the 5′ end of series of oligonucleotides composed of 10 deoxyadenosine (A) and one deoxyguanosine (G) positioned at a defined site along each strand, P-NH-CH2)n-5′-AnGA10-n3′ (n ) 2-10). Upon mixing this strand with a complementary strand composed of deoxythymidosine (T) and one deoxycytidosine (C) at the corresponding positions required for matched base pairing, 5′-T10-nCTn-3′ (n ) 2-10), a base-paired duplex was obtained. The second series employed a slightly shorter 3-aminopropyl linker between the pyrene and the 5′ edge of the oligonucleotide. In both series, the single deoxyguanosine is placed either on the pyrenyl-N-alkylbutanoamide-modified strand or on the complementary strand (Tables 1 and 2). In addition, pyrenyl-N-alkylbutanoamide was also attached (through the (CH2)6 linker) to an oligonucleotide with a randomly chosen oligonucleotide sequence that contains three deoxyguanosines
Modification and Characterization of the Oligonucleotides. Amide coupling was activated26 by using N-(3-dimethylaminopropyl)-N′-ethylcarbodimide HCl (EDC) and Nhydroxysulfosuccinimide sodium salt (sulfo-NHS), eq 1, in a DMSO/water mixture. Pyrenebutanoic acid 1 (2 mM in 0.5 mL DMSO) was stirred with EDC (Fluka, 2 mg, 9 µmol) and sulfoNHS (Fluka, 2 mg, 10 µmol) for 15 min at room temperature. This solution was added to the amino-modified oligonucleotide (Integrated DNA Technology, 200 µM in 0.5 mL aq of 0.1M Na2CO3, pH ) 8.3), and the reaction mixture was stirred for 18 h at room temperature. The product was purified on Sephadex G-25 (DNA grade, Pharmacia) and eluted with 0.01 M phosphate buffer, pH ) 7.3. The modified oligonucleotides were characterized by electro-spray and MALDI mass spectroscopy.27 The number of pyrene units per strand was measured by absorption spectrometry to be 0.8-0.9 per oligonucleotide, with the oligonucleotide concentration calculated from its absorption at 260 nm, ) 1 × 105 M-1 cm-1. (This value is calculated for an 11-mer based on an accepted approximation26 for the absorption of oligonucleotides as 104 × N (M-1 cm-1), where N is the number of bases. The extent of pyrene-N-alkylbutanoamide labeling was calculated from its absorption at 342 nm, on the basis of the measured extinction coefficient for pyrenebutanoic acid in buffered 5% DMSO: water ( ) 1.4 × 104 M-1 cm-1), Figure 1.) Whereas the pyrenyl-N-alkylbutanoamide group when attached to the oligonucleotide through the (CH2)6 linker exhibited the same absorption maximum as does free pyrenebutanoic acid in aqueous solution at 342 nm, Figure 1a, the analogous group attached similarly through the (CH2)3 linker exhibited an absorption maximum that is red-shifted to 345 nm, Figure 1b. This shift implies a slightly different environment for the pyrenyl group in the (CH2)6- and (CH2)3- linked oligonucleotides, with stronger direct interaction with the base pairs being observed with the shorter linker. The helical content of the duplexes for each series, in native or pyrene-modified form, ranged from 85% to 95% as measured by circular dichroism (CD) spectrometry (Jasco-600), Figure 2. Although a helical structure is established in each duplex by a Cotton effect at 220-300 nm,28 no signal could be observed at longer wavelengths (in the pyrenyl-N-alkylbutanoamide absorption range). There is thus no evidence for intercalation
Photophysical Quenching in Oligonucleotides
J. Phys. Chem. B, Vol. 103, No. 43, 1999 9323
TABLE 2: Rates for Fluorescence Quenching (kET) and Charge Recombination (kb) for a Series of Pyrenyl-N-hexylcarboxamide End-Labeled Oligonucleotides compound
pyrene-G distance (Å)a
kET (× 10-5 s-1)b
P-NH-(CH2)6-5′-A10-3′-3′-T10-5′ P-NH-(CH2)6-5′-A10G-3′-3′-T10C-5′ P-NH-(CH2)6-5′-A10C-3′-3′-T10G-5′ P-NH-(CH2)6-5′-A9GA-3′-3′-T9CT-5′ P-NH-(CH2)6-5′-A4GA6-3′-3′-T4CT6-5′ P-NH-(CH2)6-5′-A2GA8-3′-3′-T2CT8-5′ P-NH-(CH2)6-I‚I′d
34 34 30.6 13.6 6.8 6.8
6.1((0.2) 71((2) 76((2) 79((2) 100((2) 710((4) 1610((10)
kb (× 10-5 s-1)c 20((2) 23((2) 190((4)
a Distance was calculated between the 5′ edge of the oligonucleotide and the guanine position where each base contributes 3.4 Å.26 b kET was evaluated from the shortening in the fluorescence at (400 nm) lifetime of the excited pyrene in the duplex compared with the pyrene lifetime on a single strand of poly-A, P-NH-CH2)6-5′-A10-3′. kET ) τobs-1 - τ0-1, where τ0_) 180 ns, and τobs is the short component from the biexponential decay of the modified oligonucleotide, Table 1, entries: 3, 5, 7, 9, 11, 13, and 14. c Decay lifetimes of the pyrene radical anion were evaluated from the transient absorption spectra recorded upon laser excitation at 355 nm, 10 mJ/cm2 per pulse and with the absorption change monitored at 460 nm. d Oligonucleotides I and I′ as described in Table 1; footnote b.
Figure 1. Absorption spectra in aqueous phosphate buffer (0.01 M, pH ) 7.5) of 14 µM solutions of (a) P-NH-(CH2)6-5′-A4GA6-3′; (b) P-NH-(CH2)3-5′-A7GA3-3′.
of the appended pyrene group into the oligonucleotide grooves, either for the (CH2)6- or the (CH2)3- series. Fluorescence Quenching in (CH2)6-PyrenecarboxamideLinked Oligonucleotides. Steady-state fluorescence spectra of the pyrenyl-N-alkylbutanoamide-modified oligonucleotides are presented in Figure 3. Emission quantum yields were calculated by using 1 as a standard for fluorescence efficiency (φ ) 0.44).29 Attaching the pyrenyl-N-alkylbutanoamide group onto a polydeoxyadenosine oligonucleotide (10 mer) produces no fluorescence quenching (compared with 1), and introducing the complementary strand (10 T) as a duplex results in only a minor decrease in the observed quantum yield (to φ ) 0.43). Strong quenching of the pyrene group fluorescence was observed, however, when even a single deoxyguanosine is present, either on the modified strand or on its complement. In a single strand containing one G, the fluorescence quantum yield decreased moderately from 0.44 to 0.36, (Table 1, entries 2, 4, 8, 10, 12). This suggests weak distance dependence in a single strand, where conformational flexibility complicates the observed kinetics by allowing ET over a variety of distances. In the duplex, however, the backbone is more rigid and the distance dependence of fluorescence quenching is more evident (Table 1, entries 3, 5, 9, 11, 13). The observed fluorescence quantum yields decreased to 0.34 and 0.17 when G is present at the eleventh and the third positions from the attached pyrenyl group, respectively. Whether the deoxyguanosine quencher is present on the template strand, P-NH-(CH2)6-5′-A10G-3′ • 5′-CT103′, or on the complementary strand, P-NH-(CH2)6-5′-A10C3′ • 5′-GT10-3′, the quenching efficiency is nearly identical (Table 1, entries 5 and 7). The most efficient quenching (φ )
Figure 2. (A) Circular dichroism spectra in aqueous phosphate buffer (0.01 M, pH ) 7.5) of 3 µM solutions of (a) P-NH-(CH2)6-5′-A2GA8-3′•5′-T8CT2-3′, (b) P-NH-(CH2)6-5′-A4GA6-3′•5′-T6CT4-3′, (c) P-NH-(CH2)6-5′-A9GA1-3′•5′-T1CT9-3′, (d) unmodified oligonucleotide duplex 5′-A10G-3′•5′-CT10-3′. (B) Circular dichroism spectra in aq phosphate buffer (0.01 M, pH ) 7.5) of 3 µM solutions of (a) P-NH-(CH2)3-5′-A1GA9-3′•5′-T9CT1-3′; (b) P-NH-(CH2)3-5′-A3GA7-3′•5′-T7CT3-3′; (c) P-NH-(CH2)3-5′-A7GA3-3′•5′-T3CT7-3′; (d) unmodified oligonucleotide duplex 5′-A10G-3′•5′-CT10-3′.
0.13) was observed for the oligonucleotide, P-NH-(CH2)65′-I-3′ • 5′-I′-3′, where three deoxyguanosines are present (Table 1, entry 14). This result requires some interaction between the distant deoxyguanosines and the excited pyrene moiety and may indicate sequential electron hopping to the second and/or third deoxyguanosine. If such multiple hops were to occur, charge recombination by back electron transfer would be suppressed through a random walk that moves the transferred electron away from the site of excitation.
9324 J. Phys. Chem. B, Vol. 103, No. 43, 1999
Figure 3. Fluorescence spectra in deaerated aqueous phosphate buffer (0.01 M, pH ) 7.5) of 10 µM solutions of (a) pyrenebutyric acid; (b) P-NH-(CH2)6-5′-A10-3′•5′-T10-3′; (c) P-NH-(CH2)6-5′-A10G-3′; (d) P-NH-(CH2)6-5′-A10G-3′•5′-CT10-3′; (e) P-NH-(CH2)6-5′-A2GA83′•5′-T8CT2-3′.
Figure 4. Time-resolved emission of oligonucleotides (25 µM) in deaerated aqueous phosphate buffer (0.01 M, pH ) 7.5): (a) P-NH(CH2)6-5′-A9GA1-3′•5′-T1CT9-3′; (b) P-NH-(CH2)6-5′-A4GA6-3′•5′T6CT4-3′; and (c) P-NH-(CH2)6-5′-A2GA8-3′•5′-T8CT2-3′. λexc ) 355 nm, 10 mJ/cm2 per pulse, λem ) 400 nm.
Time-Resolved Fluorescence Quenching in (CH2)6-pyrenen-alkylbutanoamide-Linked Oligonucleotides. The observed fluorescence lifetimes for the (CH2)6′-pyrene-linked oligonucleotides reveal the same trend. On a single strand, the observed decay is fit by a dominant, but poorly resolved, single exponential with a lifetime of 90 ns when G is at third base, 120 ns when G is at the fourth or tenth position, 144 ns at the eleventh position, and 180 ns with no G on the strand (Table 1, entries 12, 10, 8, 4 and 2). This deceptively simple kinetic form likely reflects similar contributions from a broad array of differentially random-coil folded chains, both from the sixcarbon linker and the single-strand oligonucleotide. In a duplex lacking G in either strand, P-NH-(CH2)6-A10 • 5′-T10-3′, the observed fluorescence decay is monoexponential, and again the excited state exhibits a shorter lifetime than in the single A10 strand, P-NH-(CH2)6-5′-A10-3′, 162 ns and 180 ns, respectively. In the corresponding duplexes, the fluorescence decays are best fit by a well-resolved biexponential profile in which the short-lived component dominates the fit (80% - 90%), Table 1, entries 3, 5, 9, 11, 13, and Figure 4. The observed short lifetimes range from 14 ns when G is at the third position to 79 ns when G is at the eleventh position. At first, it seems surprising that quenching by deoxythymidosine is so difficult to observe experimentally, given the
Zahavy and Fox
Figure 5. Transient absorption spectra of oligonucleotides in deaerated aqueous phosphate buffer (0.01 M, pH ) 7.5) of solutions of (a) P-NH-(CH2)6-5′-A10G•5′-CT10-3′, 50 µM; and (b) P-NH-(CH2)65′-A10-3′•5′-T10-3′, 30 µM, with spectral gain of a factor of 3. λexc ) 355 nm, 10 mJ/cm2 per pulse.
observation of relatively efficient quenching by this base when pyrene was attached to a deoxyuridine and incorporated into the nucleotide sequence.30 The kinetic profiles observed here, as well as in other systems reported by Netzel,6 differ from that described for the deoxyuridine-attached pyrene and suggest that the local helix is likely perturbed significantly when the pyrenyl group is bound through a three-carbon tether (see below). When interstrand hydrogen bonding is significant, the possibility of proton-assisted electron transfer from C or T might become possible. Our system with a flexible six-carbon tether is less likely to disrupt local structure, and is therefore less likely to participate in hydrogen-bonded conformations in which deoxycytosine and deoxythymidosine evidently function as effective quenchers. In the absence of such interactions, deoxyguanosinemediated oxidative quenching of the attached pyrene becomes the dominant (monoexponential) process observed. The fluorescence lifetimes for the duplexes are shorter than the corresponding single strands, presumably because of welldefined interactions over fixed distances, since in the helical duplex structure each deoxyguanosine is fixed at a defined position along the oligonucleotide. For the pyrene-modified oligonucleotide that contains three deoxyguanosines, the observed emission decay is fit by a monoexponential with an even shorter lifetime of 6 ns (Table 1, entry 14). Transient absorption spectra of one pair of pyrene-modified oligonucleotides were recorded in order to determine whether the observed pyrene fluorescence quenching takes place through ET. Figure 5 compares the transient spectrum of an A10 duplex capped with a deoxyguanosine (P-NH-(CH2)6-5′-A10G-3′ • 5′-CT10-3′, Figure 5a) with that of the analogous duplex lacking the end-bound deoxyguanosine, P-NH-(CH2)6-5′-A10-3′ • 5′T10-3′ (Figure 5b). In the absence of deoxyguanosine (Figure 5b), the spectrum exhibits a maximum at 430 nm and a transient with a lifetime longer than 10 µs, which is assigned to a pyrenyln-alkylbutanoamide triplet.22b,31 In the presence of even a distant deoxyguanosine, however, the observed absorption maximum at 465 nm (Figure 6a) is assigned to the absorption of a substituted pyrene radical anion.22a Although the observed spectrum may at first glance appear to resemble the superimposition of spectra of a pyrene triplet and a pyrene cation radical, the short lifetimes observed for these species are incompatible with this interpretation, and the observed shift is exactly as would be predicted by the attachment of an N-alkybutanoamide substituent in a polar protic solvent. Indeed, the pyrene radical anion spectrum is known to be broadened in water with
Photophysical Quenching in Oligonucleotides
Figure 6. Time-resolved absorption spectra in deaerated aqueous phosphate buffer (0.01 M, pH ) 7.5) of 50 µM solutions of (A) P-NH-(CH2)6-5′-A2GA8-3′•5′-T8CT2-3′; and (B) P-NH-(CH2)65′-A10G-3′•5′-CT10-3′. λexc ) 355 nm, 10 mJ/cm2 per pulse, λabs ) 460 nm.
characteristics that correspond closely to those shown in Figure 6.20 The rise time for the appearance of the substituted pyrene radical anion (as measured by time-resolved absorption spectroscopy at 460 nm) is 7 ns for the duplex with G at the third position from the point of attachment (P-NH-(CH2)6-5′A2GA8-3′ • 5′-T8CT23′), Figure 6a. The reduced species then decays in 52((2) ns. When a deoxyguanosine is present in the eleventh position in P-NH-(CH2)6-5′-A10G-3′ • 5′-CT10-3′, the lifetime has increased to 470((20) ns, Figure 6b, while retaining the same spectral features. Although both thermodynamic arguments and the transient spectrum of the pyrenyl-N-alkylbutanoamide radical anion imply that distance-dependent pyrene fluorescence quenching involves ET, it is difficult to rule out completely other contributors to the quenching. Indeed, the relatively low fluorescence yields for the bases themselves may argue for rapid relaxation deriving from the proximity of the π, π* and n, π* states in the bases.32 Moreover, as discussed above, vibronically coupled internal conversion through a lower lying n, π* state is plausible and could induce pyrene group quenching in the duplexes without producing an observable transient. It is also difficult to rule out completely the production of a pyrenyl radical cation, which would overlap significantly with the observed spectrum of the pyrene radical anion. The absence of a persistent species after the decay of the radical anion suggests, however, that this route must be a minor one, if it occurs at all. We infer that since direct absorption evidence for the expected guanine radical cation G+• could not be detected on the microsecond time scale, and that fast
J. Phys. Chem. B, Vol. 103, No. 43, 1999 9325 deprotonation probably converts any of the latter species to its neutral form, G•(-H+), which has a broad absorption (400700 nm) with an extinction coefficient ( < 2000 M-1 cm-1)20c too low for detection with our apparatus. Assuming that the observed quenching is dominated by photoinduced ET oxidation of the pyrenyl group (producing a reduced deoxyguanosine), the rates for ET quenching (kET) and the back recombination (kb) can be obtained, Table 2. The fastest quenching is observed in the oligonucelotide containing three deoxyguanosines (P-NH-(CH2)6-I • I′; kET ) 1.6 × 108 s-1) where the first deoxyguanosine is on the third base and the two other deoxyguanosines are positioned further along the sequence. In the six-carbon-linked series with only one deoxyguanosine, the fastest ET is observed in P-NH-(CH2)6-5′-A2GA8-3′ • 5′-T8CT2-3′, where G is also on the third base (kET ) 7.1 × 107 s-1, kb ) 1.9 × 107 s-1). As the deoxyguanosine is moved to the fifth position in (P-NH-(CH2)6-5′-A4GA6-3′ • 5′T6CT4-3′), a sharp decrease in quenching efficiency is observed (kET ) 1 × 107 s-1, kb ) 2.3 × 106 s-1). Further separation results in only minor further decreases in the quenching rates. Faster quenching is observed in P-NH-(CH2)6-I • I′ than in P-NH-(CH2)6-5′-A2GA8-3′ • 5′-T8CT2-3′, where in both cases the closest deoxyguanosine is on the third base (but with two more deoxyguanines along the strand in the first case). The presence of the additional deoxyguanosines on the chain clearly influences the rate of fluorescence quenching and slows the rate of back electron transfer so significantly that it cannot be directly observed. This can be explained either by either an unusual (unprecedented, to our knowledge) shift of the redox potential of one reduced deoxyguanosine by another in the chain or by a more likely suppression of back ET in the multiply substituted chain, possibly through sequential electron hopping between neighboring deoxyguanosines. A plot of the variation of the rate of fluorescence quenching with distance in this series is nonlinear. For a simple electron transfer over a rigid backbone, a linear dependence would have been predicted according to eq 2, where d is the distance between the 5′ edge of the oligonucleotide and the deoxyguanosine quencher calculated by assuming that each base contributes an average of 3.4 Å to the distance.33
kET ) A • exp(-β(d - d0))
(2)
This nonlinearity suggests a complexity incompatible with a simple through-π-stacked electron transfer to deoxyguanosine being the only operative quenching route. As has been found in previous work,6,18 some much slower quenching by deoxythymidosine and/or deoxycytidosine must transpire. In the (CH2)6-linked oligonucleotide lacking a G, P-NH-(CH2)65′-A10′, introduction of the complementary strand, 5′-T10′, results in only negligible quenching of pyrenyl group emission, with the fluorescence quantum yield decreasing to 0.43 (from 0.44 in the single strand), and the fluorescence lifetime decreasing to 162 from 180 ns, yielding a very slow electron-transfer rate (kET ) 6.1 × 105 s-1). By analogy with previous work, we conclude that some other process must contribute to the observed quenching. Literature precedent may suggest that deoxythymidosines present in the complementary chain may be responsible for the slower quenching of the excited pyrene group. Nevertheless, our system differs in several ways from the system described in previous work.18,19 The pyrene group is not attached directly to the deoxythymidosine base in our oligonucleotides but rather is covalently attached through a flexible linker to a phosphate group at the 5′ end. Such a long spacer undoubtedly allows the pyrenyl group substantial con-
9326 J. Phys. Chem. B, Vol. 103, No. 43, 1999
Zahavy and Fox This efficient quenching of the excited pyrene group by deoxythymidosine and/or deoxyguanosine in the three-carbonlinked series contrasts with our observations in the six-carbonlinked series. Although the CD spectra in this series show only a weak Cotton effects at wavelengths longer than 320 nm that would indicate pyrene intercalation into the oligonucleotide grooves, a red shift of the pyrene absorption in the (CH2)3linked series, Figure 1b, implies a stronger direct interaction in the (CH2)3-linked series than in the (CH2)6-linked series between the attached pyrene and the component bases of the duplex, possibly because of the reduced conformational flexibility in the shorter spacer. Conclusions
Figure 7. Time-resolved emission in dearated aqueous phosphate buffer (0.01 M, pH ) 7.5) of 10 µM solutions of (a) P-NH-(CH2)3A10 and (b) P-NH-(CH2)3-A10•T10 (10µM). λexc ) 347 nm, 10 mJ/ cm2 per pulse, λem ) 400 nm.
TABLE 3. Fluorescence Quantum Yields and Lifetimes for a Series of Pyrenyl-N-propylcarboxamide End-Labeled Oligonucleotides compound
fluorescence quantum yield/(φ)
τslow/(ns)a ((4)
P-NH-(CH2)3-5′-A10-3′ P-NH-(CH2)3-5′-A10-3′-3′-T10-5′ P-NH-(CH2)3-5′-A7GA3-3′-3′-T7CT3-5′ P-NH-(CH2)3-5′-A3GA7-3′-3′-T3CT7-5′ P-NH-(CH2)3-5′-A1GA9-3′-3′-T1CT9-5′
0.46((0.02) 0.064((0.004) 0.064((0.004) 0.087((0.004) 0.092((0.004)
160 (100%) 147 (23%) 156 (23%) 158 (28%) 170 (30%)
a The relative contribution of the slow decays of the biexponential transient emission is presented in parentheses. The major contribution was a fast decay (< 1 ns), which was not resolved from the excitation pulse.
formational flexibility outside the duplex helix. Because the pendent pyrenyl group in our system is not intercalated in the duplex, the helix is more stable than in systems in which the chromophore intercalates within the DNA grooves. In our systems, deoxythymidosine could in principle participate in quenching assisted by internal secondary proton transfer. Whatever its role, its function as an excited-state electron acceptor is observed only when direct oxidative electron transfer to a deoxyguanosine residue is slow because it is so far down the chain from the excited pyrenyl group. Fluorescence Quenching in (CH2)3-pyrenyl-N-alkylbutanoamide-Linked Oligonucleotides. Time-resolved fluorescence of the duplexes in the three-carbon-linked series reveal biexponential decay profiles that include an initial fast quenching (τ < 1 ns) that accounts for 70-80% of the observed decay and a second slower decay taking place over a time scale of 150-170 ns and contributing about 20-30% of the decay, Figure 7. The short fluorescence lifetimes are independent of the presence or position of the deoxyguanosine quencher and are identical, within experimental error, to the decay profile observed for the duplex P-NH-(CH2)3-5′-A10-3′‚5′-T10-3′ that lacks a deoxyguanosine quencher, Table 3. These results are fully consistent with our fluorescence quantum yield measurements, where the introduction of a complementary strand composed mostly of deoxythymidosines causes the steady-state fluorescence emission efficiency to decrease by 80-90%.
The fluorescence of end-labeled oligonucleotides bearing a substituted pyrenylbutanoamide group connected through a sixcarbon-linker is quenched, likely by photoinduced electron transfer, when a deoxyguanosine is present in the oligonucleotide sequence. The deoxyguanosine can be present either along a single strand or on the same or complementary strand in a duplex. The rates of fluorescence quenching obtained by monitoring the decay in the intensity of the transient pyrene radical anion show a nonmonotonic decrease with increasing deoxyguanosine-pyrene distance. This nonlinearity implies conformational complexity and weaker quenching by some other component. The reduction of the excited pyrene moiety by deoxythymidosine in the presence of a protic solvent is the most plausible alternative quenching mode. Faster quenching is observed with oligonucleotides bearing multiple deoxyguanosines. Redox quenching by deoxyadenosine and deoxythymidosine is less efficient in this series. The unexpected dependence of deoxythymidosine quenching on local structure implies sensitivity to hyrdogen-bonding for efficient reductive quenching. When the pyrenyl group is attached to an oligonucleotide through a shorter linker (an n-propyl chain), an efficient quenching of pyrene fluorescence is observed. Helix conformational effects are significant and any such quenching contributions from vibronically coupled internal conversion show only weak structural dependence on the position of deoxyguanosine. Acknowledgment. This work was supported by the U.S. Dept. of Energy. E.Z. is grateful for a Fulbright postdoctoral fellowship that made possible his stay in Austin. References and Notes (1) (a) Purugganan, D.; Kumar, C. V.; Turro, N. J.; Barton, J. K. Science 1986, 241, 1645-1649. (b) Barton, J. K.; Kumar, C. V.; Turro, N. J. J. Am. Chem. Soc. 1986, 108, 6391-6393. (c) Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.; Bossmann, S. H.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025-1029. (d) Arkin, M. R.; Stemp, E. D. A.; Holmlin, R. E.; Barton, J. K.; Ho¨rmann, A.; Olson, E. J. C.; Barbara, P. F. Science 1996, 273, 475-480. (e) Holmlin, R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem., Int. Ed. Engl. 1997, 36, 2715-2730. (2) Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114, 36563660. (3) Fukui, K.; Tanaka, K. Angew. Chem., Int. Ed. Engl. 1998, 37, 158160. (4) (a) Hall, D. B.; Holmlin, R. E.; Barton, J. K. Nature 1996, 382, 731-735. (b) Kelley, S. O.; Holmlin, R. E.; Stemp, E. D. A.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 9861-9870. (5) Meggers, E.; Kusch, D.; Spichty, M.; Wille, U.; Giese, B. Angew. Chem., Int. Ed. Engl. 1998, 37, 460-462. (6) (a) Netzel, T. L.; Nafisi, K.; Zhao, M.; Lenhard, J. R.; Johnson, I. J. Phys. Chem. 1995, 99, 17936-17947. (b) Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L.; Chan, C.-K. J. Am. Chem. Soc. 1989, 111, 7226-7232. (c) Netzel, T. L.; Zhao, M.; Nafisi, K.; Headrick, J.; Sigman, M. S.; Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 9119-9128. (d)
Photophysical Quenching in Oligonucleotides Netzel, T. L.; Nafisi, K.; Headrick, J.; Eaton, B. E. J. Phys. Chem. 1995, 99, 17948-17955. (7) (a) Lewis, F. D.; Zhang, Y.; Letsinger, R. L. J. Am. Chem. Soc. 1997, 119, 5451-5452. (b) Lewis, F. D.; Zhang, Y.; Letsinger, R. L. J. Org. Chem. 1997, 62, 8565-8568. (c) Lewis, F. D.; Wu, T.; Zhang, Y.; Letsinger, R. L.; Greenfield, S. R.; Wasielewski, M. R. Science 1997, 277, 673-676. (8) (a) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda, M. Chem. Commun. 1997, 1609-1610. (b) Bardea, A.; Dagan, A.; Ben-Dov, I.; Amit, B.; Willner, I. Chem. Commun. 1998, 839-840. (c) Hurley, D. J.; Tor, Y. J. Am. Chem. Soc. 1998, 120, 2194-2195. (9) (a) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yosef, G. Nature 1998, 391, 775-778. (b) Storhoff, J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (c) KorriYoussoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388-7389. (10) (a) Winkler, J. R.; Gray, H. B. Chem. ReV. 1992, 92, 369-379. (b) McLendon, G. Acc. Chem. Res. 1988, 21, 160-167. (c) Isied, S. S. Electron-Transfer Reactions: Inorganic, Organometallic, and Biological Applications; Isied, S. S., Ed.; American Chemical Society: Washington, DC, 1997; Vol. 253, pp 331-348. (d) Isied, S. S.; Ogawa, M. Y.; Wishart, J. W. Chem. ReV. 1992, 92, 381. (e) Zahavy, E.; Willner, I. J. Am. Chem. Soc. 1996, 118, 12499-12514. (f) Johnson, M. K.; King, R. B.; Kurtz, D. M., Jr.; Kutal, C.; Norton, M. L.; Scott, R. A. Electron Transfer in Biology and the Solid State; Johnson, M. K.; King, R. B.; Kurtz, D. M., Jr.; Kutal, C.; Norton, M. L.; Scott, R. A., Ed.; American Chemical Society: Washington, DC, 1990. (11) Diederichsen, U. Angew. Chem., Int. Ed. Engl. 1997, 36, 23172319. (12) Olson, E. J. C.; Hu, D.; Ho¨rmann, A.; Barbara, P. F. J. Phys. Chem. B 1997, 101, 299-303. (13) Netzel, T. L. J. Chem. Educ. 1997, 74, 646-651. (14) Gasper, S. M.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 12762-12771. (15) Nakatani, K.; Shirai, J.; Sando, S.; Saito, I. J. Am. Chem. Soc. 1997, 119, 7626-7635. (16) Meggers, E.; Kusch, D.; Spichty, M.; Wille, U.; Giese, B. Angew. Chem., Int. Ed. 1998, 37, 460-462. (17) Lim, E. C. J. Phys. Chem. 1986, 90, 6770. (18) Wassam, W. A., Jr.; Lim, E. C. Chem. Phys 1980, 48, 299. (19) Clark, L. B. J. Am. Chem. Soc. 1977, 99, 3934. (20) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617618. (b) Steenken, S.; Telo, J. P.; Novais, H. M.; Candeias, L. P. J. Am.
J. Phys. Chem. B, Vol. 103, No. 43, 1999 9327 Chem. Soc. 1992, 114, 4701-4709. (c) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1989, 111, 1094-1099. (21) The reduction potential of excited pyrenebutanoic acid was evaluated from the standard redox values of pyrene, Py•+/Py ) +1.28 V, Py/Py•- ) -2.09 V, and from the 0,0 band of the modified bound pyrene. Hence, the potential of excited pyrene is *Py/Py•- ) +1.32 V. (22) (a) O’Connor, D.; Shafirovich, V. Y.; Geacintov, N. J. Phys. Chem. 1994, 98, 9831-9839. (b) Shafirovich, V. Y.; Levin, P. P.; Kuzmin, V.; Thorgeirsson, T. E.; Kliger, D. S.; Geacintov, N. E. J. Am. Chem. Soc. 1994, 116, 63-72. (c) Shafirovich, V. Y.; Courtney, S. H.; Ya, N.; Geacintov, N. E. J. Am. Chem. Soc. 1995, 117, 4920-4929. (23) Mann, J. S.; Shibata, Y.; Meehan, T. Bioconj. Chem. 1992, 3, 554558. (24) (a) Fox, M. A. Acc. Chem. Res. 1992, 25, 569-574. (b) Stewart, G. M.; Fox, M. A. Chem. Mater. 1998, 860, 0-863. (c) Stewart, G. M.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 4354-4360. (d) Watkins, D. M.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 4344-4353. (e) Fossum, R. D.; Fox, M. A. J. Am. Chem. Soc. 1997, 119, 1197-1207. (f) Fossum, R. D.; Fox, M. A. J. Phys. Chem. 1997, 101, 6384-6393. (25) (a) Galoppini, E.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 22992300. (b) Fox, M. A.; Galoppini, E. J. Am. Chem. Soc. 1997, 119, 52775285. (26) Hermanson, G. H. Bioconjugate Techniques; Academic Press: San Diego, 1996. (27) The calculated mass for P-NH-C6-5′-A10G-3′: 3855 (or 3916 with another phosphate group): Found in electro-spray mass spectrometry for the triple charged ion: 1304.4((0.4) converts to parent mass of 3912((2). Calculated mass for P-NH-C3-5′-A10G-3′: 3813 (or 3874 with another phosphate group): Found in MALDI: 3874((4) in electro-spray mass spectrometry for the triple charged ion: 1249((1) which is 3747((3) for the parent. (28) (a) Guschlbauer, W. Nucleic Acid Structure: An Introduction; Springer-Verlag: New York, 1976; Chapter 2. (b) Fasman, G. D. Circular Dichroism and the Conformational Analysis of Biomolecules; Plenum Press: New York, 1996. (29) Lianos, P.; Lux, B.; Gerard, D. J. Chim. Phys. 1980, 77, 907912. (30) Manoharan, M.; Tivel, K. L.; Zhao, M.; Nafiai, K.; Netzel, T. L. J. Phys. Chem. 1995, 99, 17461-17472. (31) Carmichael, I.; Hug, J. J. Phys. Chem. Ref. Data 1986, 15, 1-6. (32) Callis, P. R. Am. ReV. Phys. Chem. 1983, 34, 329. (33) Saenger, W. Principles of Nucleic Acid Structure; SpringerVerlag: New York, 1984.
