Fluorescence intensity decays of 2-aminopurine solutions: lifetime distribution approach

Abstract

The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

Figure 1

1a. 2AP emission spectra in neat and binary solvents of dioxane and water. Left to right are spectra of 2AP solutions in dioxane, dioxane:water (90:10) and water alone. 1b. Dependence of 2AP fluorescence intensity on the percentage of water in dioxane:water mixtures (n=3, Error = Mean ± SD). Fluorescence intensity increases with polarity resulting from addition of water in the mixture.

Figure 1

1a. 2AP emission spectra in neat and binary solvents of dioxane and water. Left to right are spectra of 2AP solutions in dioxane, dioxane:water (90:10) and water alone. 1b. Dependence of 2AP fluorescence intensity on the percentage of water in dioxane:water mixtures (n=3, Error = Mean ± SD). Fluorescence intensity increases with polarity resulting from addition of water in the mixture.

Figure 2

2a. Fluorescence intensity decays of 2AP in neat and binary mixtures of dioxane and water. 2AP lifetime, as expected, is higher in water than in dioxane and dioxane:water mixtures. 2b. Deviations from the best-fits to the mono-exponential model of 2AP fluorescence intensity decays in water (top) and a dioxane:water mixture (90:10%; bottom and 80:20%; middle). Deviations are more pronounced in binary mixtures than in neat solvents because of higher heterogeneity in binary mixtures.

Figure 2

2a. Fluorescence intensity decays of 2AP in neat and binary mixtures of dioxane and water. 2AP lifetime, as expected, is higher in water than in dioxane and dioxane:water mixtures. 2b. Deviations from the best-fits to the mono-exponential model of 2AP fluorescence intensity decays in water (top) and a dioxane:water mixture (90:10%; bottom and 80:20%; middle). Deviations are more pronounced in binary mixtures than in neat solvents because of higher heterogeneity in binary mixtures.

Figure 3

Lifetime distributions (Lorentzian model) of 2AP fluorescence intensity decays in dioxane:water mixtures. Lifetime distributions are broader in highly heterogeneous mixtures (90:10% and 80:20% dioxane:water) than either in homogenous (100% and 0% water) or in less heterogeneous mixtures (98:2% dioxane:water).

Figure 4

Emission spectra of 2AP in absence and presence of 1 M acrylamide. Glycerol prevents the conformational changes in 2AP, if any, so that the effect of acrylamide can be exclusively detected.

Figure 5

5a. Fluorescence intensity decays of 2AP in presence and absence of 1 M acrylamide. Observation was made at 370 nm. 5b. Deviations from the best fits to the mono-exponential model of 2AP fluorescence intensity decays in the absence and presence of 1 M acrylamide.

Figure 5

5a. Fluorescence intensity decays of 2AP in presence and absence of 1 M acrylamide. Observation was made at 370 nm. 5b. Deviations from the best fits to the mono-exponential model of 2AP fluorescence intensity decays in the absence and presence of 1 M acrylamide.

Figure 6

Lifetime distributions (Lorentzian model) of 2AP intensity decays in presence and absence of 1M acrylamide. Addition of 1 M acrylamide broadens the lifetime distribution (FWHM) from 2.6 ns, χ_R²=1.08 in 0 M acrylamide to 3.3 ns, χ_R²=1.13 in 1 M acrylamide, owing to increased microenvironmental heterogeneity in 1 M acrylamide solution.

Figure 7

Deviation from the best fits to the mono-exponential model of 2AP fluorescence intensity decays for various emission wavelengths. These measurements were done in glycerol at room temperature either at the long emission wavelength of 410 nm (top panel) or at short emission wavelength of 350 nm (bottom panel).

Figure 8

Lifetime distribution (Lorentzian model) of 2AP fluorescence intensity decays for various emission wavelengths. These measurements were done in glycerol at room temperature either at the long emission wavelength of 410 nm (R state) or at short emission wavelength of 350 nm (F state). 2AP solution at 350 nm is more heterogeneous (FWHM = 3.2 ns, τ₀ = 7.89 ns, χ_R²=1.53) than at 410 nm (FWHM = 1.1 ns, τ₀ = 9.14 ns, χ_R² =1.01).

Figure 9

9a. Synthetic RNA hairpin molecule “HP21” in folded state, labeled with 2AP at 21^st position. 9b. Fluorescence intensity decays of folded versus denatured RNA. 9c. Top. Residuals from multi-exponential analysis (3 exponential fit, χ_R² = 0.99), Bottom. Residuals from Lorentzian lifetime distribution analysis (unimodal Lorentzian fit, χ_R²= 1.03).