RNA has the ability to carry out a large variety of functional roles while only consisting of four different building blocks . Many structural studies have demonstrated that RNA can dramatically change its structure upon binding to a small molecule, peptide or protein, suggesting that intrinsic dynamics may be a way to expand the functional potential of RNA beyond its limited chemical repertoire. Paradigmatic examples of such behavior are observed in the HIV-1 TAR RNA, and the target DNA sequence for HhaI Methyltransferase. In both cases motions and conformational changes are known to occur and are critical for function.
Nuclear Magnetic Resonance (NMR) spectroscopy is uniquely suited to study motions in biological systems1. I employ solution NMR techniques because of their ability to probe multiple sites of a molecule at the same time. Specifically I use the techniques of Relaxation as well as Residual Dipolar Couplings (RDCs), to probe the motions of free and bound forms of RNA over the different timescales.
By definition, relaxation refers to the return of a perturbed nuclear spin population to its thermodynamic equilibrium state. As a result, it can be measured by forcing a population of molecules into a non-equilibrium state and monitoring their return to equilibrium11. Observing different time points allows for the extrapolation of the relaxation time or its inverse, the relaxation rate, and since these rates can be related back to motion, they can be used to observe the inherent dynamics.
Although relaxation experiments have the ability to reveal information over several timescales, they are unable to probe the ns-μs timescale. In addition, relaxation experiments alone are incapable of yielding information on motional trajectories. Thus I also use RDCs in combination with a motional model (3D-GAF), to extract order parameters (relative flexibility values) and motional amplitudes/trajectories for many different sites in nucleic acids. This technique provides a detailed description of the average motions present over the entire ms-ps timescale.
Briefly explained, this entails measuring inherently motionally averaged RDCs via NMR, and then fitting these values to an equation describing the system as if it were static. In order to fit these motionally averaged values to a static equation three terms describing the three dimensional motional amplitudes are included.