J. Phys. Chem. B 2006, 110, 26327-26336
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Excited-State Properties and Transitions of Fluorescent 8-Vinyl Adenosine in DNA Cyril A. Kenfack,*,†,‡ Alain Burger,§ and Yves Me´ ly† Photophysique des Interactions Biomole´ culaires, UMR 7175 CNRS, Institut Gilbert Laustriat, Faculte´ de Pharmacie, UniVersite´ Louis Pasteur, Strasbourg 1, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France, Centre de Physique Atomique Mole´ culaire et Optique Quantique, Faculte´ des Sciences, UniVersite´ de Douala, BP 8580, Douala, Cameroon, Laboratoire de Chimie Bioorganique, UMR 6001 du CNRS, UniVersite´ de Nice, Sophia Antipolis Parc Valrose, 06108 Nice, Cedex 2, France ReceiVed: July 12, 2006; In Final Form: October 13, 2006
8-Vinyl-adenosine (8VA) is a new fluorescent nucleoside analogue with improved spectroscopic properties as compared to 2-aminopurine (2AP). To further understand its photophysics, we analyzed by the timedependent density functional theory and the configuration interaction single method, the electronic properties, and transitions of 8VA in its free form and stacked with one or two flanking bases. For free 8VA, the predicted excited-state energy gaps, absorbance peak position, and oscillator strength were found to be in excellent agreement with the experimentally determined ones. Moreover, its high fluorescence quantum yield was found to be associated with the dipole-allowed S1 f S0 transition. Stacking of 8VA with C, T, or A in dimers or trimers resulted in fluorescence quenching through mechanisms that depend on the nature and relative orientation of the flanking base(s). When 8VA is stacked with T, quenching mainly results from nonradiative relaxation to low-lying dark excited state(s) that do not exist in free 8VA. When 8VA is stacked with A, quenching results mainly from mixing of the molecular orbitals in the ground state. Both types of quenching are thought to accompany the stacking of 8VA with C. In addition, the C-8VA-C trimer was found to exhibit a low-lying S1 emissive state that may generate an increased fluorescence quantum yield and lifetime. The predicted photophysical properties of the trimers are highly consistent with the spectroscopic data of a series of 15-mer oligonucleotides differing only by the nature of the residues flanking the central 8VA.
Introduction The new fluorescent nucleoside analogue 8-vinyl adenosine (8VA)1 has been reported to exhibit improved spectroscopic properties with respect to 2-aminopurine (2AP), the reference nucleoside analogue for probing local nucleic acid structure and dynamics.2-7 Its molar absorption coefficient is about twice that of 2AP, enabling a more sensitive detection. Moreover, 8VAlabeled oligonucleotides (ODNs) give more stable duplexes than the corresponding 2AP-labeled sequences when the fluorescent analogue is flanked with adenines or thymines, indicating that 8VA is less perturbing than 2AP.1 This is probably due to the vinyl group of 8VA that improves base stacking when exposed to the major groove and thus contributes to the duplex stability.8,9 However, the fluorescence of 8VA is strongly quenched in single- and double-stranded DNA, although its quantum yield drops less than the 2AP one, when incorporated in a given ODN sequence. In addition, although the fluorescence of free 8VA decays with a single lifetime of 4.7 ns, four components ranging from 53 ps to 4.39 ns1 are needed to describe its fluorescence decay in the context of DNA sequences. A similar behavior was observed with 2AP and is associated with a distribution of local conformations in which 2AP experiences varying degrees of stacking with its nearest neighbors.10,11In these stacked states, the quenching of 2AP can be static due to electron delocalization in the ground state with weak oscillator strength and/or dynamic through nonradiative relaxation to a charged transfer state (CT).1 * Corresponding author. E-mail:
[email protected]; fax: 00237 342 6710; phone: 00237 6031051. † Universite ´ Louis Pasteur. ‡ Universite ´ de Douala. § Universite ´ de Nice.
In this context, by analogy with 2AP, the interpretation of the fluorescence properties of 8VA-labeled ODNs in terms of structure and dynamics needs to determine how the electronic properties of 8VA are affected by the stacking with the neighboring bases. To reach this objective, we investigated the electronic structure of 8VA alone and stacked with thymine, cytosine, and adenine in B-form dimers and trimers, using the Configuration Interaction Singles (CIS)12,13 method and the Time-Dependent Density Functional Theory (TDDFT).14-16 These two methods were found to describe, the electronic transitions in conjugated systems satisfactorily.10,11,17-27 Our calculations allow us to assign the S0 f S1 and S0 f S2 transitions of free 8VA to π-π* and n-π* transitions, respectively. The calculated positions of the absorbance maximum, oscillator strength, and fluorescence Stokes shift of free 8VA were found to be in excellent agreement with the experimental data. As for 2AP, both static and dynamic mechanisms explain the strong quenching of 8VA stacked with its nearest neighbors in ODNs. Although similar quenching mechanisms are inferred for the two nucleobases, significant quantitative differences appear when corresponding dinucleotides and trinucleotides are compared. Finally, calculations on 8VA trimers were found to correlate well with the experimental data of a series of 15-mer ODNs differing by the nature of the residues flanking the central 8VA.1 Materials and Methods Gaussian 0328 was used for TDDFT and CIS calculations. All reported results were obtained using the triple-ζ 6-311+G(d) basis set29,30 with the hybrid B3LYP exchange-correlation function for TDDFT calculation and the triple-ζ 6-311++G(d)
10.1021/jp064388h CCC: $33.50 © 2006 American Chemical Society Published on Web 12/07/2006
26328 J. Phys. Chem. B, Vol. 110, No. 51, 2006
Kenfack et al.
basis set in the Hartree Fock scheme for CIS. The two methods express the wave function of a given excited state as a linear combination of singly excited determinants. A molecular orbital (MO) ΨI for the Ith excited state has the form
ΨI )
CijIΦifj ∑ ∑ i∈occ j∈virt
where Φifj is the determinant representing an excitation from the occupied MO φi toward the virtual MO φj, and CijI is the weight of that determinant in the wavefunction ΨI. Thus, the analysis of the nature of the states was supported by the values of the CijI coefficient in the Hartree-Fock wavefunction for CIS (TDB3LYP for TDDFT) and by the shape of the MOs. The molecular orbitals are drawn using the program Molden.31 The geometry of the free 8VA and the 8VA DNA oligomers in the ground state was obtained in INSIGHT II32 from canonical B-form X-ray structures, mutating adenine to 8VA and removing the sugar and phosphate atoms. Since substitution of A by 8VA was shown to marginally affect the stability and thus the structure of the DNAs,1 no further optimization of the base geometry was done, and no symmetry constraints were used. A counterpoise correction method was not used in this work to avoid basis set superposition error. In the case of 2AP, this error was
