Dynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solution

Adenosine-5'-monophosphate disodium salt (AMP), 2-aminopurine (2AP) and cytidine-5'-monophosphate disodium salt (CMP) were purchased from Sigma. Deoxythymidine-5'-monophosphate disodium salt (dTMP) was purchased from Abcam. Guanosine-5'-monophosphate disodium salt (GMP) was purchased from Acros Organics. These were all used as received.

HPLC grade water (Fisher) was used as the solvent throughout. Measurements were made in phosphate buffer (0.1 M, pH 7) and Tris-HCl buffer (20 mM Tris-HCl, 60 mM NaCl, 0.1 mM Na₂EDTA).

Samples of 2AP at a fixed concentration (∼3 μM) and varying concentrations of nucleotides were prepared in either phosphate or Tris-HCl buffer.

Steady-state fluorescence measurements were performed on a Horiba Fluoromax-P photon-counting spectrofluorimeter and the resulting spectra were analysed using Origin graphing software. An excitation wavelength of 325 nm was used and emission intensity was corrected for variation in the excitation lamp intensity. The detector response was constant over the emission wavelength range used. Emission intensities were determined by integration over a 180-nm bandwidth encompassing the emission maximum, and were averaged over five measurements.

Fluorescence lifetimes were measured using time-correlated single photon counting on an Edinburgh Instruments spectrometer equipped with TCC900 photon counting electronics. The excitation source was the third harmonic of the pulse-picked output of a mode-locked Ti-Sapphire laser (Coherent Mira pumped by Coherent Verdi), consisting of pulses of ∼150 fs duration at a repetition rate of 4.75 MHz and a wavelength of 310 nm. Fluorescence emission was detected orthogonal to the excitation beam through a polarizer set at the magic angle with respect to the vertically polarized excitation. A band-pass of 10 nm was used in the emission monochromator and photons were detected by a cooled microchannel plate detector (Hamamatsu R3809 series). The instrument response of the system was ∼80 ps full-width at half-maximum. Fluorescence decay curves were recorded on a time scale of 50 ns, resolved into 4096 channels, to a total of 10,000 counts in the peak channel. Decay curves were analyzed by iterative re-convolution, assuming a multi-exponential function, equation (1), using Edinburgh Instruments software FAST.

$$\begin{eqnarray}&&I\left(t\right)=\displaystyle \sum _{i=1}^{n}{A}_{i}\exp \left(\displaystyle \frac{-t}{{\tau }_{i}}\right)\end{eqnarray} \tag{ 1 }$$

where I is the fluorescence intensity as a function of time, t, (normalised to the intensity at t = 0); τ_i is the fluorescence lifetime of the ith decay component and A_i is the fractional amplitude (A-factor) of that component.

The effect of a quencher, Q, on the fluorescence quantum yield of a fluorophore, M, can be expressed by the classical Stern-Volmer equation (2) [26].

$$\begin{eqnarray}&&\displaystyle \frac{{\varnothing }_{0}}{\varnothing }=1+{K}_{{\rm{SV}}}\left[Q\right]\end{eqnarray} \tag{ 2 }$$

where ${\varnothing }_{0}$ and $\varnothing$ are the fluorescence quantum yields of M in the absence and presence of Q, respectively, [Q] is the molar concentration of Q, and ${K}_{{\rm{SV}}}\,\,$is the Stern-Volmer constant.

If quenching is purely dynamic, i.e. occurs by collision of Q with excited M, ${K}_{{\rm{SV}}}$ is equal to the dynamic quenching constant, K_D, as defined in equation (3).

$$\begin{eqnarray}&&{K}_{D}={k}_{q}{\tau }_{0}\end{eqnarray} \tag{ 3 }$$

where k_q is the bimolecular rate constant for collisional quenching and τ₀ is the fluorescence lifetime of M in the absence of the quencher.

If quenching is purely static, i.e. occurs by interaction of M and Q in the ground state to form a complex, MQ, that has a fluorescence quantum yield less than that of free M, an average quantum yield is measured, as expressed by equation (4).

$$\begin{eqnarray}&&\left\langle \varnothing \right\rangle =\alpha {\varnothing }_{0}+(1-\alpha ){\varnothing }_{{\rm{MQ}}}\end{eqnarray} \tag{ 4 }$$

where $\left\langle \varnothing \right\rangle$ is the average quantum yield in the presence of quencher, ${\varnothing }_{{\rm{MQ}}}$ is the quantum yield of MQ, and $\alpha$ is the fractional population of free M, as defined by equation (5).

$$\begin{eqnarray}&&\alpha =\,\displaystyle \frac{\left[M\right]}{\left[M\right]+\left[{\rm{MQ}}\right]}=\displaystyle \frac{1}{1+{K}_{{\rm{S}}}\left[Q\right]}\,\end{eqnarray} \tag{ 5 }$$

where K_S, is the equilibrium constant for formation of MQ (equation (6)), and is known as the static quenching constant.

$$\begin{eqnarray}&&{K}_{S}=\,\displaystyle \frac{\left[{\rm{MQ}}\right]}{\left[M\right]\left[Q\right]}\end{eqnarray} \tag{ 6 }$$

If ${\varnothing }_{MQ}$ ≪ $\varnothing$₀, equation (4) reduces to equation (7), which is identical in form to the classical Stern-Volmer expression (equation (2)), with K_SV equal to the static quenching constant, K_S. This corresponds to the commonly expressed assumption that complexes MQ are 'non-fluorescent' [26].

$$\begin{eqnarray}&&\displaystyle \frac{{\varnothing }_{0}}{\left\langle \varnothing \right\rangle }=1+{K}_{S}[Q]\end{eqnarray} \tag{ 7 }$$

If both static and dynamic quenching occur, their combined effects on the quantum yield are expressed by equation (8).

$$\begin{eqnarray}&&\left\langle \varnothing \right\rangle =\displaystyle \frac{\alpha {\varnothing }_{0}}{1+{K}_{D}[Q]}+\left(1-\alpha \right){\varnothing }_{{\rm{MQ}}}\end{eqnarray} \tag{ 8 }$$

If $\varnothing$_MQ ≪ $\varnothing$₀, this becomes the modified Stern-Volmer equation (9).

$$\begin{eqnarray}&&\displaystyle \frac{{\varnothing }_{0}}{\left\langle \varnothing \right\rangle }=\left(1+{K}_{{\rm{S}}}\left[Q\right]\right)\left(\,1+{K}_{{\rm{D}}}\left[Q\right]\right)\end{eqnarray} \tag{ 9 }$$

In steady-state fluorescence-quenching measurements, it is assumed that the ratio of the fluorescence intensity (I₀) of M in the absence of Q to that (I) in the presence of Q is equal to the corresponding quantum yield ratio. This assumption is valid provided that the intensity of excitation light absorbed by M is constant throughout the measurements.

Time-resolved fluorescence measurements permit the effects of dynamic and static quenching to be separated. Dynamic quenching is manifested by a decrease in the fluorescence lifetime of M with increasing concentration of Q, according to equation (10).

$$\begin{eqnarray}&&\displaystyle \frac{{\tau }_{0}}{{\tau }_{{\rm{M}}}}=1+{k}_{{\rm{q}}}{\tau }_{0}\left[Q\right]\end{eqnarray} \tag{ 10 }$$

where τ₀ and τ_M are the fluorescence lifetimes of M in the absence and presence of Q, respectively.

In the presence of static quenching, if the fluorescence lifetime of MQ is too short to be measurable (i.e. MQ is effectively non-fluorescent), then a mono-exponential decay will be observed with lifetime, τ_Μ. However, if the lifetime of MQ, τ_MQ, is measurable, an additional component will be seen in the fluorescence decay, as given by equation (11).

$$\begin{eqnarray}&&I\left(t\right)=\alpha \exp \left(\displaystyle \frac{-t}{{\tau }_{{\rm{M}}}}\right)+(1-\alpha )\exp \left(\displaystyle \frac{-t}{{\tau }_{{\rm{MQ}}}}\right)\end{eqnarray} \tag{ 11 }$$

where I is the fluorescence intensity as a function of time, t, (normalised to the intensity at t = 0); both α (defined in equation (4)) and τ_M (equation (10)) depend on [Q], but τ_MQ is independent of [Q].

The modified Stern-Volmer equation may then be expressed in terms of the number-average lifetime, $\left\langle \tau \right\rangle .$

$$\begin{eqnarray}&&\displaystyle \frac{{\tau }_{0}}{\left\langle \tau \right\rangle }=\left(1+{K}_{{\rm{S}}}\left[Q\right]\right)\left(\,1+{k}_{{\rm{q}}}{\tau }_{0}\left[Q\right]\right)\end{eqnarray} \tag{ 12 }$$

where $\left\langle \tau \right\rangle =\alpha {\tau }_{{\rm{M}}}+(1-\alpha ){\tau }_{{\rm{MQ}}}.$

These measurements were made under the same buffer conditions as those used by Rachofsky et al [23], but we chose to use the natural bases in the form of the NMPs, rather than the nucleosides, because of their increased solubility. Furthermore, we used an excitation wavelength of 325 nm for our steady-state measurements, to minimise the inner filter effect that might occur due to absorption by the NMPs at high concentrations. Rachofsky et al [23] used an excitation wavelength of 309 nm, assuming negligible absorbance by the natural bases at this wavelength. However, although the molar absorption coefficients of the natural bases at this wavelength are very small, there is sufficient absorbance at concentrations of 10's of mM over a path-length of 0.5 cm to significantly attenuate the excitation intensity. This effect is greatest for GMP and TMP (see figures S1 and S2 of supplementary information, available online at stacks.iop.org/MAF/8/025002/mmedia), resulting in a decrease in intensity with increasing NMP concentration equivalent to K_SV values of ∼14 M⁻¹ and ∼7 M⁻¹, respectively, and deviation from linearity at concentrations above 0.03 M.

The dependence of the fluorescence intensity of 2AP on the concentration of each of the NMPs is shown in figure 1, in the form of a classic Stern-Volmer plot. The data could be fitted adequately by equation (2) yielding the values given in table 1 for the Stern-Volmer constant, K_SV. However, there may be small deviations from linearity, suggesting the occurrence of static quenching. Rachofsky et al [23] reported only dynamic quenching constants obtained from time-resolved fluorescence measurements. They indicated that they were unable to obtain static quenching constants because of the low solubility of the ribosides, but commented that upward curvature in Stern-Volmer plots of steady-state data was observed.

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To obtain definitive dynamic quenching constants, we carried out time-resolved fluorescence measurements. An excitation wavelength of 310 nm (near the maximum of the 2AP absorption spectrum) was used, since the inner filter effect does not affect the fluorescence lifetime, only the fluorescence intensity. The decay parameters of 2AP measured as a function of GMP concentration are given in table 2.

For concentrations of GMP up to 1 mM, single exponential decays were observed, with the lifetime decreasing with increasing quencher concentration, as expected for dynamic quenching (equation (10)). However, above this concentration, an additional two, shorter, decay components (τ₂ and τ₃) appeared, which can be attributed to the onset of static quenching. The 3-component decays for GMP concentrations from 5 to 50 mM could be fitted globally with τ₂ and τ₃ as common (i.e. concentration-independent) lifetimes; the value of τ₁ decreases steadily with increasing GMP concentration. The fractional populations (A factors) of components τ₂ and τ₃ increase with increasing GMP concentration. This is consistent with a model in which τ₁ is due to free 2AP undergoing dynamic quenching by GMP, while τ₂ and τ₃ are due to two photophysically distinct 2AP-GMP complexes, in which 2AP is statically quenched by GMP. The nature of the complexes (as reflected by their lifetimes) is independent of GMP concentration, but their populations increase as GMP concentration increases. In the τ₃ complex, 2AP is highly quenched by interaction with GMP to give a lifetime of only 60 ps. This is comparable to the very short lifetimes observed for 2AP stacked with G in DNA duplexes [1] and for the 2AP-G dinucleotide [19, 27], implying that there is intimate stacking of 2AP and G in the τ₃ complex. The much longer lifetime of the τ₂ complex suggests a structure in which there is much weaker interaction between 2AP and G, but still significant quenching. The fractional population of the τ₃ complex is much greater than that of the τ₂ complex, indicating that a well-stacked structure is favoured. This is in line with computational prediction of highly stacked structures for non-covalent complexes of 2AP and the natural bases [21, 22, 28].

Similar decay behaviour was observed for 2AP in the presence of the other NMPs, as shown in tables S1–S3 in the supplementary information. In all cases, two statically-quenched complex populations are observed at concentrations above 1 mM, a minor population with a lifetime (τ₂) of ∼3 ns, and the major component with a much shorter lifetime (τ₃). The value of τ₃ depends on the identity of the natural base, and decreases in the order A > C > G ∼ T, indicating a decrease in quenching efficiency in that order. In all cases, at a NMP concentration of 50 mM, the τ₃ complex accounts for ∼30% of the emitting population, while τ₂ constitutes less than 10%. Although the statically quenched complexes show measurable fluorescence, their average quantum yield is no more than 3% that of unquenched 2AP.

The value of K_D, and hence k_q, (equation (3)) for each NMP was determined from a plot of $\tfrac{{\tau }_{0}}{{\tau }_{M}}$ versus [NMP] (equation (10)), where τ_M is the lifetime component τ₁. The linear fits are shown in figure 2 and the quenching constants are given in table 3.

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We find that the rate constant for collisional quenching, k_q, is greater for GMP and dTMP than for AMP and CMP. Our k_q values for GMP and AMP differ significantly from those of Rachofsky et al [23]; we find that GMP has the greatest value of k_q, whereas they report that guanosine has the lowest k_q value and adenosine the highest. Rachofsky et al [23] do not present the fluorescence decay data that they used to obtain k_q values; however, it is evident that they observed multi-exponential decays at higher nucleoside concentrations, and we note that they incorrectly employed the intensity-average lifetime for Stern-Volmer analysis.

The considerable difference between the K_D values obtained from lifetime data (table 3) and the Ksv values obtained from the classic Stern-Volmer plots (table 1, figure 1) confirms the occurrence of static quenching. We used three alternative methods to determine the static quenching constant for each of the NMPs: (i) fluorescence intensity data were fitted with the modified Stern-Volmer equation (equation (9)), with the value of K_D fixed; (ii) average lifetime data (tables 2 and S1–S3 of supplementary information) were fitted with the modified Stern-Volmer equation (equation (12)) with the value of K_D fixed; (iii) from linear plots of 1/α versus [NMP] (equation (5)), where α, the fractional population of free 2AP, is obtained from decay data as the value of A₁ (table 2 and S1–S3 of supplementary information). Examples of (ii) and (iii) are shown for AMP in figures 3 and 4. The data and fits for the other NMPs are given in the supplementary information (figures S3 and S4). The values of K_s obtained are given in table 4. The values of K_S obtained from the two decay-data-based methods (τ₀/〈τ〉 and 1/α) are in good agreement and are similar for all the NMPs, indicating a similar propensity for stacking with 2AP in all cases.

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However, for CMP and dTMP, there is a significant discrepancy between these values and those obtained from intensity data. This suggests that I₀/I is not a reliable measure of relative quantum yield in these cases; the quantum yield of the complexes relative to free 2AP is overestimated. This would be accounted for by a greater molar absorption coefficient of 2AP in complex with these NMPs than that of free 2AP, at the excitation wavelength of 325 nm. Since we are exciting on the red edge of the 2AP absorption spectrum, a small complexation-induced spectral shift could result in a signifcant increase in absorption coefficient compared with free 2AP. It has been observed previously that interbase stacking interactions lead to a red-shift of a few nm in the absoprtion or excitation spectrum of 2AP when it is inserted into oligonucleotides [29–31].

To replicate the conditions used by Narayanan et al [24], the quenching of the fluorescence of the 2AP base by each of the natural nucleoside monophosphates was investigated in 0.1 M phosphate buffer.

The dependence of the fluorescence intensity of 2AP on the concentration of each of the NMPs is shown in the Stern-Volmer plots in figure 5, and could be fitted adequately by equation (2), yielding the values given in table 1 for the Stern-Volmer constant, K_SV. For GMP in this buffer, we saw evidence of G-quartet formation [32] (increase in viscosity of the solution and scattering of excitation light) at concentrations above 20 mM. Consequently, the analysis of quenching was restricted to lower concentrations, and this prevented the analysis of static quenching by GMP in this buffer. The formation of G-quartets, a form of self-aggregation unique to guanosine, is enhanced in this buffer system by the high concentration of potassium ions [32].

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Narayanan et al [24] chose to fit their intensity data to the modified Stern-Volmer equation (equation (9)), with both K_D and K_S as variable parameters. They do not comment on the deviation from linearity of their data, and, since they present the data plotted on a logarithmic concentration scale, this cannot be discerned. However, we find that the functions fitted by Narayanan et al [24] can be very well approximated by linear Stern-Volmer functions (equation (2)) (R² > 0.998) over the range of concentrations measured (as shown in figure S5 of the supplementary information). This suggests that their data could be fitted adequately by the linear equation; indeed the values of K_S that they report are small (<5 M⁻¹), with relatively high errors. The K_SV values that we estimate from their results, on that basis, are given in table 1. These values are in good agreement with those of the present work, and follow the same trend.

To further investigate the quenching process, the fluorescence decay of 2AP was measured as a function of NMP concentration. We found that the fluorescence lifetime of 2AP in this buffer, in the absence of NMP, is much shorter than expected, only 5.7 ns, half that in Tris-HCl buffer (table 2). This suggested that the buffer itself (H₂PO₄⁻/HPO₄²⁻) was acting as a quencher. This was confirmed by measurement of the lifetime as a function of buffer concentration (figure S6 of supplementary information), which demonstrated dynamic quenching, with k_q = 1.00 × 10⁹ M⁻¹ s⁻¹. To our knowledge the quenching of 2AP fluorescence by phosphate buffer has not been reported previously, although phosphate anions have been observed to quench the fluorescence of tryptophan and tyrosine [33–35]. Narayanan et al [24] were unaware of the quenching effect of the buffer and erroneously used a literature value of 11.4 ns for τ₀ in calculating k_q from K_D.

For AMP, CMP and dTMP, the effect of their concentration on the fluorescence decay parameters of 2AP was similar to that observed in Tris-HCl buffer, as shown in tables S4–S6 of supplementary information. Monoexponential decays at sub-mM NMP concentrations switch to 3-exponential decays at concentrations above 1 mM, with the onset of static quenching. The values of τ₂ and τ₃ are shorter than in Tris-HCl buffer, suggesting that the interaction between 2AP and the NMP in the statically quenched complexes is influenced by the composition of the buffer. Nevertheless, the trend in τ₃, A > C > G, T, is the same as seen in Tris-HCl.

For GMP, the decay parameters (table S7 of supplementary information) are anomalous due to G-quartet formation; this is manifested mainly as very high amplitudes of the shortest decay component at concentrations above 20 mM. (This may be due to some type of enhanced quenching process, but could be an artefact due to intense light scattering by the guanosine aggregates.) Therefore, the use of these decay data was restricted to the determination of the dynamic quenching constant at concentrations ≤20 mM.

The values of K_D and k_q determined from the lifetime data are given in table 5. The Stern-Volmer plots are shown in figure 6. As a consequence of the much shorter τ₀, the K_D vaues are approximately one half those measured in Tris-HCl (table 3), but the k_q values and their trend are very similar in both buffers. The values of k_q reported by Narajanan et al [24] are in surprisingly good agreement, given that they were calculated on the assumption of a τ₀ of 11.4 ns. However, this is merely a fortuitous consequence of their substantial overestimation of the K_D values from fits of intensity data to the modified Stern-Volmer equation.

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Static quenching constants for AMP, CMP and dTMP were determined as described above, yielding the values shown in table 6. Related plots are shown in figures S7 and S8 of supplementary information. The values of K_S are comparable to those measured in Tris-HCl, although somewhat lower. Complexation appears to be less favourable in the phosphate buffer. A similar discrepancy between decay-derived and intensity-derived values is evident for CMP and dTMP. The values reported by Narayanan et al [24] are significantly less than our values and show much greater variability between the nucleosides. This, together with the overstimation of K_D values (vide supra), is symptomatic of the correlation of variables inherent in unconstrained fitting of data with the modified Stern-Volmer equation.