Hidden intermediates in Mango III RNA aptamer folding revealed by pressure perturbation

Abstract

Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.

Figure 1

Structure of the Mango III aptamer. (A and B) Two 180° orientations of the original Mango III aptamer structure (PDB: 6e8s) reported in Trachman et al. (15). Lower (dark blue) and upper (teal) G-quadruplexes are capped by dye (TOI-B, purple spheres). The dye is capped by the canonical WC A-U basepair (yellow). The G-quadruplex is stabilized by a potassium ion (large green sphere) and interacts with a flanking helix of WC basepairs (wheat). Underlying the G-quadruplex are three hydrogen-bonded bases A30-U24-A12 (light pink) followed by a canonical double helix (red). Two bases, A11 and U16 (CPK colors), are extruded from the structure, and one base, U21 (pale green), interacts with the dye. (C) Topology of the Mango aptamer structure used in the present study (color coded as in A and B), which has four extra basepairs in the bottom stem (in black), and the basepair, G6-C41 (underlined in red), in that stem is inversed compared with the crystal structure in which it is the second basepair, C2-G37.

Figure 2

¹H NMR spectrum of Mango III at 10 mM KCl. (A) 1D ¹H NMR spectra in absence of dye (yellow) and in presence of TO (blue). The most prominent peaks to appear in the G-quadruplex imino range are labeled G1–G4. (B) 2D ¹H–¹H NOE spectra in absence of dye and (C) in presence of dye. Spectra were acquired in 20 mM MOPS buffer, 20°C (pH 7.2). Several new peaks appear in the 1D spectrum, both in the G-quadruplex region (∼11–12 ppm) and in the duplex region (∼12–14 ppm) upon addition of TO and correspond to imino protons that become protected upon stabilization of the G-quadruplex by the dye. Three NOEs between G-quadruplex imino protons and three NOEs between duplex imino protons and the G-quadruplex amino/aromatic protons also appear upon TO binding. NOESY spectra were acquired using a 700-MHz spectrometer equipped with a cryoprobe.

Figure 3

Pressure dependence of Mango III TO fluorescence. (A) at 10 mM KCl 20°C; (B) at 10 mM KCl 5°C (red: increasing pressure, blue: decreasing pressure); (C) Data and fits of the increasing pressure profiles at 1 (red), 10 (blue), and 100 (green) mM KCl and 20°C.

Figure 4

Pressure dependence of the imino region of the 1D ¹H NMR spectrum of Mango III. Pressures are 10 bar (blue), 1000 bar (red), 2000 bar (green), and 3000 bar (purple). The spectrum at atmospheric pressure in absence of dye is also shown for reference (yellow). All spectra were acquired in presence of TO except at atmospheric pressure, no dye (yellow). Experimental conditions were 10 mM KCl, 20 mM MOPS (pH 7.2), 20°C.

Figure 5

Pressure dependence of the intensity of the main G-quadruplex imino proton peaks in Fig. 2. (A) G1, (B) G2, (C) G3, and (D) G4. 1 mM KCl (red), 10 mM KCl (blue), and 100 mM KCl (green).

Figure 6

Schematic for the transition pathways of Mango III deduced from the high-pressure NMR and fluorescence experiments. MIII_F is fully folded, maximally fluorescent Mango III. MIII_{GD_TOI} is Mango III in which the G-quadruplex is disrupted and the dye is bound in an intermediate fluorescent state. MIII_{GF_TOI} is Mango III in which the G-quadruplex is folded and the dye is bound in an intermediate fluorescent state. Finally, MIII_GD + TO is Mango III with disrupted G-quadruplex and free dye.