Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence

Abstract

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding-unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.

FIGURE 1

Structure of 2-AP-labeled RNA substrates. (A) Structural formulae of U:A and U:2-AP base pairs. (B) mFold-predicted structure of hairpin RNA substrates. Spheres denote the sites of specific 2-AP substitutions (6 position in red, 14 position in purple, 21 position in green). (C) Nuclease footprinting of folded hairpin substrates, comparing uncut (UC), RNase T₁-digested (T₁), and RNase T₂-digested (T₂) RNAs. The lane designated “⁻OH” is a sequence ladder generated via limited alkaline hydrolysis of HP14. The black arrow denotes RNase T₁-directed cleavage at nucleotide G12, and the bracket indicates preferential RNase T₂ cleavage sites at positions 10–15. (D) Sequence alignment of each of the 8-mer control RNAs with the hairpin substrate. 2-AP was inserted as the sixth nucleotide for each control RNA, analogous to the position of the 2-AP substitution in each corresponding full length hairpin.

FIGURE 2

Emission spectra of 2-AP-substituted RNA oligonucleotides. Fluorescence (λ_ex = 303 nm) of RNA hairpins (solid lines) and control sequences (dashed lines) was measured under native conditions (10 mM KHEPES (pH 7.4), 50 mM KOAc, 5 mM Mg(OAc)₂) at 25 °C using 300 nM of each RNA substrate.

FIGURE 3

Thermal denaturation analyses of 2-AP-substituted RNA substrates. (A) Blank-corrected fluorescence (λ_ex = 303 nm; λ_em = 370 nm) of hairpin (solid lines) and control (dashed lines) RNA substrates was measured under native conditions (10 mM KHEPES (pH 7.4), 50 mM KOAc, 5 mM Mg(OAc)₂) as a function of temperature. To improve signal-to-noise, HP14, C14, HP21, and C21 samples were measured at 750 nM with 10 nm emission slits. (B) Fluorescence intensity values from hairpin substrates were normalized to emission from cognate 8-mer control RNA sequences at each measured temperature. (C) These control-corrected hairpin emission values were then used to construct derivative plots as a function of temperature (ΔT = 4 °C), permitting estimation of transition melting temperatures (T_m) listed in Table 2.

FIGURE 4

Chemical denaturation of 2-AP-substituted RNA substrates. (A) Blank-corrected fluorescence from hairpin (solid circles) and control (open circles) RNA substrates was measured at 10 mM KHEPES (pH 7.4) across a titration of urea at 25 °C. (B) Fluorescence intensity values from hairpin substrates were normalized to emission from cognate 8-mer control RNA sequences at each urea concentration. Parameters relating the thermodynamics of urea-dependent RNA unfolding were estimated using a two-state model as described under Experimental Procedures (solid lines) and are listed in Table 2.

FIGURE 5

Magnesium-stabilized folding of hairpin RNA substrates in 4.5 M urea. The fluorescence intensities of hairpin (solid circles) and 8-mer control (open circles) RNA substrates were measured as a function of Mg²⁺ concentration at 25 °C, and plotted relative to the emission from each substrate in the absence of added Mg²⁺. Data were resolved by nonlinear regression using the cooperative Hill equation (eq. 3, solid lines). Averaged regression parameters from five independent experiments are listed in Table 3.

FIGURE 6

Time-domain intensity decay of HP6 (red) and C6 (blue) RNA substrates following excitation at 295 nm (3 µM RNA in 10 mM NaHEPES (pH 7.4), 50 mM NaOAc, 5 mM Mg(OAc)₂) at 22 °C. The instrument response function is shown in green. Residual plots from nonlinear regression of 2-AP fluorescence decay data to three component exponential series based on eq. 4 are shown below. Lifetime parameters calculated from regression series are summarized in Table 4.