Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution

Abstract

Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

Figure 1

Numerical simulations of the longitudinal relaxation behavior of the imino proton spin system in A-U (left hand figures), and G-C (right hand figures) Watson-Crick base pairs using coordinates from the TAR-TAR*_GA complex (PDB accession number 2rn1). The relaxation behavior was simulated by numerical integration of the Solomon equations for all protons of the TAR-TAR*_GA complex, and by taking into account stochastic exchange processes between the labile ¹H in the RNA and the water ¹H that was assumed to remain in thermodynamic equilibrium throughout the calculation. For all simulations, an overall tumbling correlation time of τ_c = 8 ns was assumed, and internal dynamics were neglected. The ¹H resonance frequency was set to 800 MHz. The simulations are representative of a single transient of a one-pulse NMR experiment. They do not take into account partial saturation of non-imino protons in a multi-scan experiment. (a) Chemical structure of A-U (left), and G-C (right) RNA base pairs. (b) Time evolution of the ¹H polarization for the non-imino protons, illustrating the response of those protons after selective excitation of the imino protons. The color code used for these curves corresponds to the color of the proton sites in the chemical structures of the RNA base pairs. All water-exchange rate constants were set to zero for these calculations to better visualize the relative importance of individual non-imino protons for the energy uptake from the imino protons. (c) Recovery of imino proton polarization as a function of relaxation time after selective (solid lines) or non-selective (dashed lines) ¹H excitation. Three calculations were performed in both cases assuming the following exchange rates with water hydrogens: τ_ex = 0.5 s⁻¹ for iminos, τ_ex = 1.0 s⁻¹ for hydroxyls (HO₂’), and τ_ex = 1.0 s⁻¹, 5.0 s⁻¹, and 10.0 s⁻¹ for aminos. (d) Typical chemical shift ranges for different ¹H types in RNA.

Figure 2

(a) NMR pulse sequences used to probe the relaxation behavior of imino protons for different spin excitation conditions: non-selective excitation (open squares), water-flip-back excitation (filled squares), and imino-selective excitation (filled circles). In all cases, a watergate sequence using an imino-selective 180° pulse with a REBURP shape was applied. Imino-selective excitation is achieved by a 90° PC9 pulse. All imino ¹H pulses are centered at 12.5 ppm, and covering a bandwidth of 4.0 ppm. For water flip-back, a sinc shape is used centered on the water frequency, and covering a bandwidth of ~1 ppm. The delay Δ was set to 1/(2J_NH), and a 2-step phase cycle ϕÊ=Êϕ _recÊ=Êx,−x was used, in order to detect only signals from ¹⁵N-labelled RNA. Secondary structures of the TAR- TAR*_GA complex, and the tRNA^Val are shown in panels (b) and (c), respectively. Experimental sensitivity curves as a function of scan time, T_scan, (sum of pulse sequence duration, acquisition time, and recovery delay), measured for (d) the TAR- TAR*_GA complex at 10° C, (e) the TAR- ^TAR*GA complex at 25° C, and (f) tRNA^Val at 25°C. Filled circles, filled squares, and open squares correspond to the results obtained from imino-selective, water-flip-back, and non-selective excitation schemes, respectively.

Figure 3

(a) Experimental evaluation of the performance of the ¹H-¹⁵N SOFAST-HMQC , optimized for imino groups in tRNA^Val as a function of scan time. The lower curve shows a linear interpolation of the optimal ¹H excitation flip angle for a given scan time as determined from a series of 1D SOFAST-HMQC spectra recorded with the flip angle varied from 90° to 150° (in 5° steps). The middle curve displays the SOFAST-HMQC intensity obtained using an optimized flip angle for each scan time. The upper curve shows the sensitivity gain observed for SOFAST-HMQC when compared to a standard water-flip-back HMQC experiment. (b) 2D SOFAST-HMQC spectrum of 1mM unlabeled (equivalent to 2 μM ¹⁵N-labelled) TAR- TAR*_GA at 10°C recorded in 12 hrs. The recycle delay between scans has been set to 180 ms, and the flip angle to 120°, corresponding to the conditions yielding maximal sensitivity for this RNA. (c) 2D SOFAST-HMQC spectrum of ¹⁵N-labeled tRNA^Val acquired in 3 s. A recycle delay of 1 ms, and a flip angle of 140° was used. Only 10 complex t₁ points were recorded with a reduced ¹⁵N spectral width of 550 Hz, resulting in extensive spectral aliasing but no additional peak overlap. Low-power ¹⁵N decoupling using an adiabatic WURST40 sequence (γB₁/2π ≈ 650Hz) was applied during an acquisition time of 40 ms to limit the radiofrequency load of the cryogenic probe.

Figure 4

(a) 2D ¹H-¹⁵N BEST-TROSY pulse sequence. Filled and open symbols correspond to 90° and 180° rf pulses, respectively. The carrier frequencies are set to 12.5 ppm (¹H), 150 ppm (¹³C), and 155 ppm (¹⁵N). ¹H pulses cover a bandwidth of 4 ppm, and have the following shapes and durations (at 800 MHz): (1) REBURP , 1.5 ms (δ₁), (2) PC9 , 2.2 ms (δ₂), and (3) EBURP-2 , 1.4 ms (δ3). An asterisk indicates time and phase reversal of the corresponding pulse shape. For ¹³C and ¹⁵N labeled RNA samples a hyperbolic secant pulse covering a bandwidth of 200 ppm is used for ¹³C decoupling during t₁ evolution. The transfer delays are adjusted to τ₁ =1/(4J_NH) −0.5δ₁ −0.5δ₂, τ₂ = 1/(4J_NH) −0.5δ₁ −κδ₃, and τ₃ = 1/(4J_NH) These settings account for spin evolution during the various shaped ¹H pulses. The parameter κ ≈ 0.8 can be fine tuned to equilibrate the transfer amplitudes of the different coherence transfer pathways for optimal suppression of the unwanted quadruplet components in the spectrum. Pulses are applied along the x-axis unless indicated. The phase φ₀ needs to be set to +y or −y, depending on the spectrometer, to add the signals originating from ¹H and ¹⁵N polarization . Two repetitions of the experiment need to be recorded for echo/anti-echo-type quadrature detection in the ¹⁵N dimension using the following phase cycle for the TROSY component (peak 1): (I) φ₁=−x,x, − y,y; φ₂=y φ₃=x; φ_acq= −x,x,y,−y; and (I) φ1⁼−x,x,− y,y; φ 2⁼−y φ3⁼−x; φ_acq=−x,x,−y,y. The semi-TROSY (peak 2) components of the peak quadruplet depicted in panel (b) can be selected using the same phase cycles, but adding 180° to phase φ₂ for both quadrature components. (b) Schematic representation of the peak quadruplet observed in a ¹H-¹⁵N correlation spectrum recorded without ¹⁵N decoupling in t₁ and t₂. (c) 2D BEST-TROSY (left) and BEST-semi-TROSY (right) spectra of tRNA^Val recorded at 25°C and 800 MHz. The data were acquired using the pulse scheme shown in (a) with a recycle delay of 200 ms. A total of 200 complex points were acquired in the t₁ dimension for a ¹⁵N spectral width of 30 ppm, resulting in acquisition times of 15 minutes per spectrum. (d) 1D traces extracted at the ¹H chemical shift of residues G1 and U64 from the 2D spectra shown in (c), where the ¹H-¹⁵N coupling constants extracted from the relative peak positions measured in the 2 spectra are shown.

Figure 5

BEST-HNN-COSY pulse sequence (a) for the detection of N-H···N hydrogen bonds in RNA. The same ¹H pulse shapes, transfer delays τ₁, τ₂, and τ₃, and carrier frequencies presented in figure 4 are used here, with the exception that the ¹⁵N carrier is switched from 155 ppm to 185 ppm between time points a and b. A trans-hydrogen-bond transfer delay 2Δ =Ê30 ms was used. A composite 59.4°_x−298°_−x−59.4°_x pulse is applied for ¹⁵N refocusing in the middle of each transfer period 2Δ. The following 4-step phase cycle is used: φ₁=x,y,−x,−y; φ₂=y,−x,−y,x; φ₃=−y,x, y,−x;φ_acq=x,−y,−x,y. States-type quadrature detection in t₁ is achieved by 90° phase incrementation of φ₁ and φ₂. (b) BEST-HNN-COSY spectrum of tRNA^Val, acquired at 25°C and 800 MHz with a recycle delay of 200 ms. A total of 200 complex points were recorded in the t₁ dimension for a ¹⁵N spectral width of 10000 Hz recorded in 20 min. For each hydrogen-bonded imino ¹H, a pair of correlations is detected (dashed lines). A histogram shows the average sensitivity gain (and standard deviation) for individual peak groups obtained by comparing the BEST-HNN-COSY with the conventional HNN-COSY experiment of Dingley et al. , performed with a recycle delay of 1.5 s for optimal sensitivity.