Characterizing Slow Chemical Exchange in Nucleic Acids by Carbon CEST and Low Spin-Lock Field R_1ρ NMR Spectroscopy

Abstract

Quantitative characterization of dynamic exchange between various conformational states provides essential insights into the molecular basis of many regulatory RNA functions. Here, we present an application of nucleic-acid-optimized carbon chemical exchange saturation transfer (CEST) and low spin-lock field R1ρ relaxation dispersion (RD) NMR experiments in characterizing slow chemical exchange in nucleic acids that is otherwise difficult if not impossible to be quantified by the ZZ-exchange NMR experiment. We demonstrated the application on a 47-nucleotide fluoride riboswitch in the ligand-free state, for which CEST and R1ρ RD profiles of base and sugar carbons revealed slow exchange dynamics involving a sparsely populated (p ~ 10%) and shortly lived (τ ~ 10 ms) NMR “invisible” state. The utility of CEST and low spin-lock field R1ρ RD experiments in studying slow exchange was further validated in characterizing an exchange as slow as ~60 s−1.

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

“…Techniques based on spin relaxation in the rotating frame (R1ρ) 1–3 and chemical exchange saturation transfer (CEST) 4–6 are providing rare insights into short-lived (μs-ms) low-populated (<5%) excited states (ESs) 7 in nucleic acids that play important roles in gene expression and regulation 8,9 . For example, canonical Watson-Crick A–T and G–C base pairs (bps) in duplex DNA have been shown to exist in dynamic equilibrium with non-canonical Hoogsteen bps 10 (Figure 1A).…”

Evaluating the uncertainty in exchange parameters determined from off-resonance R1ρ relaxation dispersion for systems in fast exchange

“…[11] The exchange timescale accessible to R1ρ is broader than CPMG (~60 s −1 < k ex < ~100,000 s −1 )[11] and for slow-intermediate exchange, the sign of excited state chemical shift sign can deduced at a single magnetic field strength[12]. For processes occurring at even slower timescales (~20 s −1 < k ex < ~300 s −1 ) chemical-exchange saturation transfer (CEST) experiments employing weak RF spin lock fields have recently been shown to be a robust approach to characterize lowly populated conformational states in both proteins[13, 14] and nucleic acids[15]. …”

“…[9, 19] While CPMG relaxation dispersion is widely used in studies of proteins, R1ρ methods are often applied to uniformly 13 C/ 15 N labeled nucleic acids because unwanted carbon-carbon interactions can be more easily suppressed. [15, 22–25] Because higher effective fields can be used in the R1ρ experiment as compared to CPMG, it is also better suited for characterizing faster microsecond exchange processes. [11, 26]…”

Effects of J couplings and unobservable minor states on kinetics parameters extracted from CEST data

“…Protein dynamics has been an increasingly interesting area with growing awareness that protein stays in more than one conformations [1][2][3][4][5][6][7][8][9]. Large biomolecules such as protein which usually present in multiple forms are themselves exchanging systems, in which there exist dynamic equilibriums between differently populated forms of the molecules.…”

“…The situation has been improving over the past half century with the development of new biophysical methods, including new nuclear magnetic resonance (NMR) techniques. Experimental methods for quantifying chemical exchange by NMR mainly includes R 1q relaxation dispersion [8,10], Carr Purcell Meiboom Gill (CPMG) relaxation dispersion [6,7,11], longitudinal magnetization exchange, and line shape analysis [12,13]. CPMG relaxation dispersion experiments, developed in the past half of a century, are a powerful technique for intermediate exchange processes (approximately 200-2000 s À1 ) [6].…”