Single-Molecule Three-Color Fret Reveals Multi-State Conformational Dynamics Of Rna Four-Way Junctions

Non-coding RNAs are involved in many essential processes in the cell such as protein synthesis in the ribosome and gene regulation in the form of riboswitches. The function of these catalytic RNAs is linked to their ability to form unique tertiary structures, which is inherently dynamic and dependent on interactions with proteins or small molecule ligands such as magnesium ions. Here, we apply three-color single-molecule FRET to study the global structural dynamics of helical RNA junctions, one of the most common structural building blocks of RNA. Three-color FRET offers the possibility to monitor three distances simultaneously, allowing one to address complex coordinated motions within single molecules. The method is applied to the unrestrained RNA four-way junction of the hairpin ribozyme. By labeling three of the four helices with fluorescent dyes, we can monitor their movement and determine their relative position. Quantitative information about the three-dimensional distribution of inter-dye distances is obtained by three-color photon distribution analysis (3C-PDA), allowing us to characterize the static conformations of the RNA four-way junction at high magnesium concentrations. To study the dynamics of the junction at lower concentrations of magnesium, we extended the toolbox of single-molecule FRET experiments, consisting of qualitative indicators of dynamics and the quantitative methods of filtered fluorescence correlation spectroscopy (fFCS) and dynamic PDA, for three-color FRET experiments. Specifically, in three-color pulsed interleaved excitation experiments (PIE) with multiparameter fluorescence detection (MFD), the multidimensional information of color, lifetime and anisotropy offers superior contrast to separate the different conformational states. This increases the robustness of the analysis and the sensitivity to minor conformational states, which are otherwise hidden in two-color FRET experiments. The experimental results are supported by enhanced sampling molecular dynamics simulations that probe the conformational space of the junction.