2-Aminopurine fluorescence quenching and lifetimes: Role of base stacking

Abstract

2-Aminopurine (2AP) is a fluorescent analog of guanosine and adenosine and has been used to probe nucleic acid structure and dynamics. Its spectral features in nucleic acids have been interpreted phenomenologically, in the absence of a rigorous electronic description of the context-dependence of 2AP fluorescence. Now, by using time-dependent density functional theory, we describe the excited-state properties of 2AP in a B-form dinucleotide stacked with guanosine, adenosine, cytosine, or thymine. Calculations predict that 2AP fluorescence is quenched statically when stacked with purines, because of mixing of the molecular orbitals in the ground state. In contrast, quenching is predicted to be dynamic when 2AP is stacked with pyrimidines, because of formation of a low-lying dark excited state. The different quenching mechanisms will result in different experimentally measured fluorescence lifetimes and quantum yields. The nucleotide 2-aminopurine (2AP) has been used as a site-specific probe of nucleic acid structure and dynamics (1-11) because it base pairs with cytosine in a wobble configuration (4,5,12) or with thymine in a Watson-Crick geometry (7,11). Thermodynamic measurements of DNAs containing 2AP:C or 2AP:T show that the former pairing is more destabilizing (11,12). Incorporating 2AP into DNA quenches its fluorescence (2, 8, 11), reducing its quantum yield from that of the free nucleoside (0.65 in aqueous solution). This reduction is attributed to stacking interactions with nearest neighbor nucleobases, and, therefore, fluorescence properties of 2AP have been used to probe the equilibrium stacking properties of DNA duplexes containing these mismatched pairs (2,8,10). Although the fluorescence decay of 2AP in solution is single exponential, with a lifetime of Ϸ10 ns, in the context of a DNA molecule, four decay components, from 50 ps to 8 ns, are typically needed to describe its lifetime (2). The short decay time observed for 2AP in a duplex is attributed to the fully stacked state, whereas the longest lifetime comes from unstacked 2AP (2, 7). The distribution of stacked states can be altered by temperature, solvent, flanking bases, and bound protein, and so all of these conditions can be probed by using 2AP fluorescence. Dynamics of stacked bases adjacent to a mutagenic mismatch in DNA (such as 2AP:C and 2AP:T) may be part of the recognition mechanism by replication͞repair enzymes (3, 13), so interpretation of 2AP fluorescence data is critical for describing these environments.To understand the effect of base stacking on fluorescent properties of 2AP, we have used time-dependent density functional theory (TDDFT) (14, 15) to calculate the excited-state properties of 2AP alone and in stacked B-and A-form dinucleotides (dimers). Analysis of monomer calculations indicates the accuracy with which TDDFT describes spectral properties: excited-state transition energies, transition dipole directions, and oscillator strengths for 2AP, thymine, cytosine, adenine, and guanine (see figures) are in excellent agreement w...

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations

“…As shown in Figure 3, left panel, 2AP fluorescence intensity at position P7 increases with temperature from 4°C-35°C. These data are consistent with a structural model in which the stacking of P7 is disrupted at higher temperatures; quenching of its fluorescence due to formation of an electronic "supermolecule" state (Jean and Hall 2001) is progressively diminished as the 2AP spends less of its time stacked. In contrast, 2AP fluorescence emission at P8 has its highest intensity at 4°C, and becomes progressively less with increasing temperature (Fig.…”

“…The sum of the amplitudes equals the apparent r 0 anisotropy value, which here varies from 0.29 to 0.33; the theoretical maximum value of r 0 for free 2AP is 0.4. Larsen et al (2001) attribute similar r 0 values observed in 2AP-DNA experiments to solvent relaxation, but based on electronic structure calculations (Jean andHall 2001, 2002), interactions with other bases could lead to very rapid depolarization that lower the apparent initial value.…”

“…A short lifetime is anticipated in the P7 decay, because when P7 is stacked over C6:G10 (even though this stacking is not canonical Aform geometry), stacking of the aromatic bases is sufficient to form an electronic "supermolecule" where electronic states of the bases are mixed. This construct will lead to very rapid fluorescence decay ( ) through charge transfer to the neighboring base (Wan et al 2000;Jean and Hall 2001). Fluorescence emission decay components as short as 9-20 psec (Larsen et al 2001) and 30-70 psec (Guest et al 1991) have been measured for 2AP in a stacked conforma-tion.…”

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

“…Studies have shown that aromatic stacking of AP with other bases gives rise to static quenching of fluorescence and that base collisions give rise to dynamic fluorescence quenching (28,29). Static quenching is sensitive to the local equilibrium structure of a DNA and may arise due to reversible formation of a ground-state binary complex between the fluorophore and the quencher.…”

“…Similar shifts in fluorescent maxima have been reported for PhIP (32) and benzo[a]pyrene (33) when these carcinogens are stacked with the bases at the lesion site. The shifts might be a result of universal (dipole-dipole relaxation) and/or specific (formation of H-bonds or other type of stacking) interactions in the excited state between the fluorophore and the neighboring group during fluorescence lifetime (28,34). A recent study involving quantum mechanical calculations of the (AP)T dinucleotide suggests that this shift arises from a perturbed excited-state electronic structure of AP resulting from a stacked conformation (28).…”