A Slow Dynamic RNA Switch Regulates Processing of microRNA-21.

The microRNAs are non-coding RNAs which post-transcriptionally regulate the expression of many eukaryotic genes, and whose dysregulation is a driver of human disease. Here we report the discovery of a very slow (0.1 s-1) conformational rearrangement at the Dicer cleavage site of pre-miR-21, which regulates the relative concentration of readily- and inefficiently-processed RNA structural states. We show that this dynamic switch is affected by single nucleotide mutations and can be biased by small molecule and peptide ligands, which can direct the microRNA to occupy the inefficiently processed state and reduce processing efficiency. This result reveals a new mechanism of RNA regulation and suggests a chemical approach to suppressing or activating pathogenic microRNAs by selective stabilization of their unprocessed or processed states.

On The Potential of Dynamic Nuclear Polarization Enhanced Diamonds in Solid-State and Dissolution (13) C†NMR Spectroscopy.

Dynamic nuclear polarization (DNP) is a versatile option to improve the sensitivity of NMR and MRI. This versatility has elicited interest for overcoming potential limitations of these techniques, including the achievement of solid-state polarization enhancement at ambient conditions, and the maximization of (13) C signal lifetimes for performing in vivo MRI scans. This study explores whether diamond's (13) C behavior in nano- and micro-particles could be used to achieve these ends. The characteristics of diamond's DNP enhancement were analyzed for different magnetic fields, grain sizes, and sample environments ranging from cryogenic to ambient temperatures, in both solution and solid-state experiments. It was found that (13) C NMR signals could be boosted by orders of magnitude in either low- or room-temperature solid-state DNP experiments by utilizing naturally occurring paramagnetic P1 substitutional nitrogen defects. We attribute this behavior to the unusually long electronic/nuclear spin-lattice relaxation times characteristic of diamond, coupled with a time-independent cross-effect-like polarization transfer mechanism facilitated by a matching of the nitrogen-related hyperfine coupling and the (13) C Zeeman splitting. The efficiency of this solid-state polarization process, however, is harder to exploit in dissolution DNP-enhanced MRI contexts. The prospects for utilizing polarized diamond approaching nanoscale dimensions for both solid and solution applications are briefly discussed.

Intermediate rate atomic trajectories of RNA by solid-state NMR spectroscopy.

Many RNAs undergo large conformational changes in response to the binding of proteins and small molecules. However, when RNA functional dynamics occur in the nanosecond-microsecond time scale, they become invisible to traditional solution NMR relaxation methods. Residual dipolar coupling methods have revealed the presence of extensive nanosecond-microsecond domain motions in HIV-1 TAR RNA, but this technique lacks information on the rates of motions. We have used solid-state deuterium NMR to quantitatively describe trajectories of key residues in TAR by exploiting the sensitivity of this technique to motions that occur in the nanosecond-microsecond regime. Deuterium line shape and relaxation data were used to model motions of residues within the TAR binding interface. The resulting motional models indicate two functionally essential bases within the single-stranded bulge sample both the free and Tat-bound conformations on the microsecond time scale in the complete absence of the protein. Thus, our results strongly support a conformational capture mechanism for recognition: the protein does not induce a new RNA structure, but instead captures an already-populated conformation.

Hydration dependent dynamics in RNA.

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in (2)H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state (13)C relaxation measurements, we establish that ns-micros motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.

Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR.

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 A decrease in the separation between phosphorothioate and 2'F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR-tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P-19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA-peptide complex.

Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR.

We have performed solid-state 31P-19F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA2'FUTGGC)*d(GCCAAT(pS)TT GCG) (A3T2) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the 2'FU7-pS19 inter-label distance, was 9.4 +/- 0.7 A, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 A decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 A, consistent with the results of a previous solution NMR study of the 2:1 complex. These 31P-19F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7-14 A in DNA-drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.