Base-Stacking Heterogeneity in RNA Resolved by Fluorescence-Detected Circular Dichroism Spectroscopy

Abstract

RNA plays a critical role in many biological processes, and the structures it adopts are intimately linked to those functions. Among many factors that contribute to RNA folding, van der Waals interactions between adjacent nucleobases stabilize structures in which the bases are stacked on top of one another. Here, we utilize fluorescence-detected circular dichroism spectroscopy (FDCD) to investigate base-stacking heterogeneity in RNA labeled with the fluorescent adenine analogue 2-aminopurine (2-AP). Comparison of standard (transmission-detected) CD and FDCD spectra reveals that in dinucleotides, 2-AP fluorescence is emitted almost exclusively by unstacked molecules. In a trinucleotide, some fluorescence is emitted by a population of stacked and highly quenched molecules, but more than half originates from a minor ∼10% population of unstacked molecules. The combination of FDCD and standard CD measurements reveals the prevalence of stacked and unstacked conformational subpopulations as well as their relative fluorescence quantum yields.

Figure 1

(A) Watson–Crick base pairs between adenine and uracil (left) and 2-AP and uracil (right). Chemical modification is indicated in red. (B and C) Absorbance (left) and fluorescence emission (right) spectra of the samples used in this study in aqueous phosphate buffer (B) or phosphate buffer containing 30% ethanol (C). Absorbance spectra have been scaled so that the longest-wavelength absorbance peak has a value of 2 for 2-AP dinucleotide (orange) and 1 for all other samples. Fluorescence spectra report the intensity per 2-AP residue and have been scaled such that free 2-AP riboside in aqueous buffer has a peak intensity of 1.

Figure 2

(A) Schematic of 2-AP dinucleotide in a stacked conformation (top) in which it is highly quenched and a brighter unstacked conformation (bottom). (B) Spectra of 2-AP riboside. (C) Spectra of 2-AP dinucleotide. Magenta, FDCD spectra processed with eq 1; green, FDCD spectra processed with eq 2; blue, standard CD spectra. (D) Magenta: FDCD spectrum of 2-AP dinucleotide processed using eq 1 under the assumption that all observed fluorescence comes from unstacked “nucleoside-like” structures. This spectrum shows strong agreement with the nucleoside FDCD (green) and CD (blue) spectra. (E) Illustration of how CD and FDCD report on different conformational subpopulations. The inhomogeneously broadened absorbance spectrum of 2-AP dinucleotide contains contributions (sticks) from stacked (blue) and unstacked (red) conformations. Labels below absorption bands indicate whether the stacked and/or unstacked conformations contribute to that band’s FDCD and/or CD signal. Nonzero CD is observed when 2-AP has optical activity, while nonzero FDCD is observed when 2-AP is fluorescent and optically active.

Figure 3

FDCD and CD spectra of (2-AP)C (A), C(2-AP) (B), and C(2-AP)C (C). Magenta, FDCD spectra processed with eq 1; green, standard CD spectra. (D) Overlay of FDCD spectra for all aqueous samples, processed with eq 1.

Figure 4

Fluorescence quenching and stacking heterogeneity in C(2-AP)C. (A) Model used for data analysis. In the unstacked (“un”) conformation, 2-AP has properties characteristic of the free nucleoside “nuc”, while the stacked conformation (“st”) has a different CD signal and fluorescence quantum yield. c = concentration, ε = extinction coefficient, Δε = ε_L – ε_R, ϕ = quantum yield. (B) Results of the “smoothed, 2 extinction coefficient” model for C(2-AP)C in aqueous buffer. Top: Close-up of the long-wavelength region of Figure 3C. Magenta, FDCD spectra processed with eq 1; green, FDCD spectra processed with eq 2; blue, standard CD spectra. Middle: Value of the quenching ratio R_ϕ obtained at each wavelength by solving the system of eqs 3 and 7. Bottom: Value of the fraction unstacked f_un = (c_tot – c_st)/c_tot obtained at each wavelength. Dashed lines indicate the wavelength range over which parameter values were quantified. (C) Analogous plots for C(2-AP)C in buffer containing 30% v/v ethanol.