Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21.

We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells. These molecules are highly ligand-efficient (MW < 330) and display specific biochemical and cellular activity by suppressing the maturation of miR-21, thereby providing an avenue toward therapeutic development in multiple diseases where miR-21 is abnormally expressed.

A novel sample handling system for dissolution dynamic nuclear polarization experiments.

We present a system for facilitated sample vitrification, melting, and transfer in dissolution dynamic nuclear polarization (DDNP) experiments. In DDNP, a sample is typically hyperpolarized at cryogenic temperatures before dissolution with hot solvent and transfer to a nuclear magnetic resonance (NMR) spectrometer for detection in the liquid state. The resulting signal enhancements can exceed 4 orders of magnitude. However, the sudden temperature jump from cryogenic temperatures close to 1 K to ambient conditions imposes a particular challenge. It is necessary to rapidly melt the sample to avoid a prohibitively fast decay of hyperpolarization. Here, we demonstrate a sample dissolution method that facilitates the temperature jump by eliminating the need to open the cryostat used to cool the sample. This is achieved by inserting the sample through an airlock in combination with a dedicated dissolution system that is inserted through the same airlock shortly before the melting event. The advantages are threefold: (1) the cryostat can be operated continuously at low temperatures. (2) The melting process is rapid as no pressurization steps of the cryostat are required. (3) Blockages of the dissolution system due to freezing of solvents during melting and transfer are minimized.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR.

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW's enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-1H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.

Natural Abundance, Single-Scan 13C-13C-Based Structural Elucidations by Dissolution DNP NMR.

While 13C-based Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) experiments offer an attractive alternative for establishing molecular structures, they suffer from low sensitivities arising from the scarcity of spin pairs present at natural abundance. Herein we demonstrate that dissolution dynamic nuclear polarization (dDNP) provides sufficient sensitivity to acquire 1D 13C INADEQUATE spectra in a single scan and at natural abundance. Moreover, if steps are adopted to endow sub-Hertz precision to these measurements, they allow one to measure carbon-carbon J couplings over both one and multiple bonds for each chemical site. As these JCC-couplings are usually sufficiently distinct to enable univocal pairing of the nuclei involved, essentially the same information as in 2D INADEQUATE can be obtained. The feasibility of the method is demonstrated for a range of compounds, including natural products such as α-pinene, menthol and limonene. Features and extensions of this approach are briefly discussed.

Sensitivity-enhanced detection of non-labile proton and carbon NMR spectra on water resonances.

Chemical exchange saturation transfer (CEST) experiments enhance the NMR signals of labile protons by continuously transferring these protons' saturation to an abundant solvent pool like water. The present study expands these principles by fusing into these experiments homonuclear isotropic mixing sequences, enabling the water-enhanced detection of non-exchangeable species. Further opportunities are opened by the addition of coupling-mediated heteronuclear polarization transfers, which then impose on the water resonance a saturation stemming from non-labile heteronuclear species like 13C. To multiplex the ensuing experiments, these relayed approaches are combined with time-domain schemes involving multiple Ramsey-labeling experiments imparting the frequencies of the non-labile sites on the water resonance, via chemical exchange. 13C and 1H NMR spectra were detected in this fashion with about two-fold SNR amplification vis-à-vis conventionally detected spectroscopies. When combined with non-uniform sampling principles, this methodology thus becomes a sensitive alternative to detect non-exchangeable species in biomolecules. Still, multiple parameters including the scalar couplings and solvent exchange rates, will affect the efficiency and consequently the practicality of the overall experiment.

Heteronuclear 1D and 2D NMR Resonances Detected by Chemical Exchange Saturation Transfer to Water.

A method to detect NMR spectra from heteronuclei through the modulation that they impose on a water resonance is exemplified. The approach exploits chemical exchange saturation transfers, which can magnify the signal of labile protons through their influence on a water peak. To impose a heteronuclear modulation on water, an HMQC-type sequence was combined with the FLEX approach. 1D 15 N NMR spectra of exchanging sites could thus be detected, with about tenfold amplifications over the 15 N modulations afforded by conventionally detected HMQC NMR spectroscopy. Extensions of this approach enable 2D heteronuclear acquisitions on directly bonded 1 H-15 N spin pairs, also with significant signal amplification. Despite the interesting limits of detection that these signal enhancements could open in NMR spectroscopy, these gains are constrained by the rates of solvent exchange of the targeted heteronuclear pairs, as well as by spectrometer instabilities affecting the intense water resonances detected in these experiments.

Acquiring and processing ultrafast biomolecular 2D NMR experiments using a referenced-based correction.

Thanks to their special spatiotemporal encoding/decoding scheme, ultrafast (UF) NMR sequences can deliver arbitrary 2D spectra following a single excitation. Regardless of their nature, these sequences have in common their tracing of a path in the [Formula: see text]-[Formula: see text] plane, that will deliver the spectrum being sought after a 1D Fourier transformation versus [Formula: see text]. This need to simultaneously digitize two domains, tends to impose bandwidth limitations along all spectral axes. Along the [Formula: see text]/[Formula: see text] dimension this problem is exacerbated by the fact that odd and even time points are not equispaced, and by additional artifacts such as time shifts between time points sampled while under the action of positive and negative decoding gradients. As a result, odd and even [Formula: see text] points are typically Fourier transformed separately, halving the potential spectral width along this dimension. While this halving of the [Formula: see text] span can be overcome by an interlaced Fourier transform, this post-processing is seldom used because of its sensitivity to hardware inaccuracies requiring even finer corrections of the even/odd [Formula: see text] data points. These corrections have so far been done manually, but are challenging to implement when dealing with low signal-to-noise ratio signals like those associated with biomolecular NMR experiments. This study introduces an algorithm for an automatic correction of all even/odd ultrafast NMR inconsistencies, based on the acquisition of a reference scan on the solvent. This algorithm was verified experimentally using an [Formula: see text]-[Formula: see text] UF-HSQC variant on ubiquitin at 600 MHz. Features of this method as well as of the interlaced Fourier transformation in general, are discussed.

Nanoporous frameworks exhibiting multiple stimuli responsiveness.

Nanoporous frameworks are polymeric materials built from rigid molecules, which give rise to their nanoporous structures with applications in gas sorption and storage, catalysis and others. Conceptually new applications could emerge, should these beneficial properties be manipulated by external stimuli in a reversible manner. One approach to render nanoporous frameworks responsive to external signals would be to immobilize molecular switches within their nanopores. Although the majority of molecular switches require conformational freedom to isomerize, and switching in the solid state is prohibited, the nanopores may provide enough room for the switches to efficiently isomerize. Here we describe two families of nanoporous materials incorporating the spiropyran molecular switch. These materials exhibit a variety of interesting properties, including reversible photochromism and acidochromism under solvent-free conditions, light-controlled capture and release of metal ions, as well reversible chromism induced by solvation/desolvation.

Toward single-shot pure-shift solution 1H NMR by trains of BIRD-based homonuclear decoupling.

Achieving homonuclear 1H decoupling remains one of the key challenges in liquid-state NMR. Such spectra would endow a variety of organic and analytical applications with an increased resolution, and would ideally do so even in a one-dimensional format. A number of parallel efforts aimed at achieving this goal using two-dimensional acquisitions have been proposed; approaches demonstrated over recent years include, among others, new modes for achieving purely-absorptive J spectroscopy, the use of spatially-selective manipulations, and exploiting the natural spin dilution afforded by heteronuclei. The present study relies on the latter approach, and explores the use of BIRD pulses distinguishing between protons bonded to (13)C from those bonded to (12)C, to achieve homonuclear decoupling in a continuous 1D scan. Studies on several representative compounds demonstrate that this goal can be implemented in a robust format, provided that suitable care is also taken to suppress unwanted coherences, of making all manipulations sufficiently broad-banded, and to provide adequate heteronuclear decoupling of the targeted protons. Dependable homonuclear decoupling performance can then be achieved, with minimal line width, fine-tuning, and sensitivity penalties.

Monitoring mechanistic details in the synthesis of pyrimidines via real-time, ultrafast multidimensional NMR spectroscopy.

Recent years have witnessed unprecedented advances in the development of fast multidimensional NMR acquisition techniques. This progress could open valuable new opportunities for the elucidation of chemical and biochemical processes. This study demonstrates one such capability, with the first real-time Two-dimensional (2D) dynamic analysis of a complex organic reaction relying on unlabeled substrates. Implementing such measurements required the development of new ultrafast 2D methods, capable of monitoring multiple spectral regions of interest as the reaction progressed. The alternate application of these acquisitions in an interleaved, excitation-optimized fashion, allowed us to extract new structural and dynamic insight concerning the reaction between aliphatic ketones and triflic anhydride in the presence of nitriles to yield alkylpyrimidines. Up to 2500 2D NMR data sets were thus collected over the course of this nearly 100 min long reaction, in an approach resembling that used in functional magnetic resonance imaging. With the aid of these new frequency-selective low-gradient strength experiments, supplemented by chemical shift calculations of the spectral coordinates observed in the 2D heteronuclear correlations, previously postulated intermediates involved in the alkylpyrimidine formation process could be confirmed, and hitherto undetected ones were revealed. The potential and limitations of the resulting methods are discussed.

Theory of nonrigid rotational motion applied to NMR relaxation in RNA.

Solution NMR spectroscopy can elucidate many features of the structure and dynamics of macromolecules, yet relaxation measurements, the most common source of experimental information on dynamics, can sample only certain ranges of dynamic rates. A complete characterization of motion of a macromolecule thus requires the introduction of complementary experimental approaches. Solid-state NMR spectroscopy successfully probes the time scale of nanoseconds to microseconds, a dynamic window where solution NMR results have been deficient, and probes conditions where the averaging effects of rotational diffusion of the molecule are absent. Combining the results of the two distinct techniques within a single framework provides greater insight into dynamics, but this task requires the common interpretation of results recorded under very different experimental conditions. Herein, we provide a unified description of dynamics that is robust to the presence of large-scale conformational exchange, where the diffusion tensor of the molecule varies on a time scale comparable to rotational diffusion in solution. We apply this methodology to the HIV-1 TAR RNA molecule, where conformational rearrangements are both substantial and functionally important. The formalism described herein is of greater generality than earlier combined solid-state/solution NMR interpretations, if detailed molecular structures are available, and can offer a more complete description of RNA dynamics than either solution or solid-state NMR spectroscopy alone.

Deuterium magic angle spinning NMR used to study the dynamics of peptides adsorbed onto polystyrene and functionalized polystyrene surfaces.

LKα14 is a 14 amino acid peptide with a periodic sequence of leucine and lysine residues consistent with an amphipathic α-helix. This "hydrophobic periodicity" has been found to result in an α-helical secondary structure at air-water interfaces and on both polar and nonpolar solid polymer surfaces. In this paper, the dynamics of LKα14 peptides, selectively deuterated at a single leucine and adsorbed onto polystyrene and carboxylated polystyrene beads, are studied using (2)H magic angle spinning (MAS) solid state NMR over a 100 °C temperature range. We first demonstrate the sensitivity enhancement possible with (2)H MAS techniques, which in turn enables us to obtain high-quality (2)H NMR spectra for selectively deuterated peptides adsorbed onto solid polymer surfaces. The extensive literature shows that the dynamics of leucine side chains are sensitive to the local structural environment of the protein. Therefore, the degree to which the dynamics of leucine side chains and the backbone of the peptide LKα14 are influenced by surface proximity and surface chemistry is studied as a function of temperature with (2)H MAS NMR. It is found that the dynamics of the leucine side chains in LKα14 depend strongly upon the orientation of the polymer on the surface, which in turn depends on whether the LKα14 peptide adsorbs onto a polar or nonpolar surface. (2)H MAS line shapes therefore permit probes of surface orientation over a wide temperature range.

Slow exchange model of nonrigid rotational motion in RNA for combined solid-state and solution NMR studies.

Functional RNA molecules are conformationally dynamic and sample a multitude of dynamic modes over a wide range of frequencies. Thus, a comprehensive description of RNA dynamics requires the inclusion of a broad range of motions across multiple dynamic rates which must be derived from multiple spectroscopies. Here we describe a slow conformational exchange theoretical approach to combining the description of local motions in RNA that occur in the nanosecond to microsecond window and are detected by solid-state NMR with nonrigid rotational motion of the HIV-1 transactivation response element (TAR) RNA in solution as observed by solution NMR. This theoretical model unifies the experimental results generated by solution and solid-state NMR and provides a comprehensive view of the dynamics of HIV-1 TAR RNA, a well-known paradigm of an RNA where function requires extensive conformational rearrangements. This methodology provides a quantitative atomic level view of the amplitudes and rates of the local and collective displacements of the TAR RNA molecule and provides directly motional parameters for the conformational capture hypothesis of this classical RNA-ligand interaction.

A REDOR study of diammonium hydrogen phosphate: a model for distance measurements from adsorbed molecules to surfaces.

Magic angle spinning NMR techniques can be used to determine the molecular structure of proteins adsorbed onto polymer and mineral surfaces, but the degree to which the orientation of proteins on surfaces can be uniquely determined by NMR is less well understood. In this manuscript, REDOR data obtained from model systems are analyzed with a view to determine the orientation of rare spins coupled to a lattice populated by strongly coupled spin 1/2 nuclei. When the surface is populated by closely spaced spins, the REDOR dephasing of a rare spin on the protein contact point to the surface is under certain circumstances complicated by contributions from homonuclear dipolar interactions between the spins of the lattice. To study multiple spin effects on the dephasing signal in rotational-echo-double-resonance experiments, we carried out a measurement on crystalline diammonium hydrogen phosphate as a model for a spin system with multiple dipolar interactions. Information about the (31)P-(31)P interactions is gathered from the reference measurement in the experiment. To fit the experimental (15)N and (31)P dephasing data well, it was necessary to account for as many as 6 and 8 spins in simulations, respectively. Using a single spin-pair interaction with an unknown distance yielded a good fit to the (31)P data with a distance of 2.7A that is nearly an Angström shorter than the shortest distance in the crystal structure. Homonuclear couplings are shown to have a significant effect on the expected dephasing.