Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

RNA Dynamics by NMR Spectroscopy.

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined-Lego like-than in proteins, hence the numerous revelations of RNA excited states.

Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy.

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy. Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution-state in-cell NMR spectroscopy (e.g., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.

Efficient Detection of Structure and Dynamics in Unlabeled RNAs: The SELOPE Approach.

The knowledge of structure and dynamics is crucial to explain the function of RNAs. While nuclear magnetic resonance (NMR) is well suited to probe these for complex biomolecules, it requires expensive, isotopically labeled samples, and long measurement times. Here we present SELOPE, a new robust, proton-only NMR method that allows us to obtain site-specific overview of structure and dynamics in an entire RNA molecule using an unlabeled sample. SELOPE simplifies assignment and allows for cost-effective screening of the response of nucleic acids to physiological changes (e.g. ion concentration) or screening of drugs in a high throughput fashion. This single technique allows us to probe an unprecedented range of exchange time scales (the whole µs to ms motion range) with increased sensitivity, surpassing all current experiments to detect chemical exchange. For the first time we could describe an RNA excited state using an unlabeled RNA.

Comprehensive analysis of NMR data using advanced line shape fitting.

NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT ( https://pint-nmr.github.io/PINT/ ). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.

Capturing Excited States in the Fast-Intermediate Exchange Limit in Biological Systems Using 1 H†NMR Spectroscopy.

Changes in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high-energy states. In this study, we present a versatile 1 H R1ρ RD experiment that not only extends the exchange timescales at least three times beyond the rate limits of 13 C/15 N R1ρ and ten times for CPMG experiments, but also makes use of easily accessible probes, thus allowing a general description of biologically important excited states. This technique can be used to extract chemical shifts for the structural characterization of excited states and to elucidate complex excited states.

Nanostructure of Materials Determined by Relayed Paramagnetic Relaxation Enhancement.

Particle and domain sizes strongly influence the properties of materials. Here we present an NMR approach based on paramagnetic relaxation enhancement (PRE) relayed by spin diffusion (SD), which allows us to determine lengths in the nm-µm range. We demonstrate the method on multicomponent organic polymer mixtures by selectively doping one component with a paramagnetic center in order to measure the domain size in a second component. Using this approach we determine domain sizes in ethyl cellulose/hydroxypropyl cellulose film coatings in pharmaceutical controlled release formulations. Here we measure particle sizes ranging from around 50 to 200 nm.

On the tuning of high-resolution NMR probes.

Three optimum conditions for the tuning of NMR probes are compared: the conventional tuning optimum, which is based on radio-frequency pulse efficiency, the spin noise tuning optimum based on the line shape of the spin noise signal, and the newly introduced frequency shift tuning optimum, which minimizes the frequency pushing effect on strong signals. The latter results if the radiation damping feedback field is not in perfect quadrature to the precessing magnetization. According to the conventional RLC (resistor-inductor-capacitor) resonant circuit model, the optima should be identical, but significant deviations are found experimentally at low temperatures, in particular on cryogenically cooled probes. The existence of different optima with respect to frequency pushing and spin noise line shape has important consequences on the nonlinearity of spin dynamics at high polarization levels and the implementation of experiments on cold probes.

Polarizing agents for efficient high field DNP solid-state NMR spectroscopy under magic-angle spinning: from design principles to formulation strategies.

Dynamic Nuclear Polarization (DNP) has recently emerged as a cornerstone approach to enhance the sensitivity of solid-state NMR spectroscopy under Magic Angle Spinning (MAS), opening unprecedented analytical opportunities in chemistry and biology. DNP relies on a polarization transfer from unpaired electrons (present in endogenous or exogenous polarizing agents) to nearby nuclei. Developing and designing new polarizing sources for DNP solid-state NMR spectroscopy is currently an extremely active research field per se, that has recently led to significant breakthroughs and key achievements, in particular at high magnetic fields. This review describes recent developments in this area, highlighting key design principles that have been established over time and led to the introduction of increasingly more efficient polarizing sources. After a short introduction, Section 2 presents a brief history of solid-state DNP, highlighting the main polarization transfer schemes. The third section is devoted to the development of dinitroxide radicals, discussing the guidelines that were progressively established to design the fine-tuned molecular structures in use today. In Section 4, we describe recent efforts in developing hybrid radicals composed of a narrow EPR line radical covalently linked to a nitroxide, highlighting the parameters that modulate the DNP efficiency of these mixed structures. Section 5 reviews recent advances in the design of metal complexes suitable for DNP MAS NMR as exogenous electron sources. In parallel, current strategies that exploit metal ions as endogenous polarization sources are discussed. Section 6 briefly describes the recent introduction of mixed-valence radicals. In the last part, experimental aspects regarding sample formulation are reviewed to make best use of these polarizing agents in a broad panel of application fields.

MQD--multiplex-quadrature detection in multi-dimensional NMR.

With multiplex-quadrature detection (MQD) the tasks of coherence selection and quadrature separation in N-dimensional heteronuclear NMR experiments are merged. Thus the number of acquisitions required to achieve a desired resolution in the indirect dimensions is significantly reduced. The minimum number of transients per indirect data point, which have to be combined to give pure-phase spectra, is thus decreased by a factor (3/4)(N-1). This reduction is achieved without adjustable parameters. We demonstrate the advantage by MQD 3D HNCO and HCCH-TOCSY spectra affording the same resolution and the same per-scan sensitivity as standard phase-cycled ones, but obtained in only 56 % of the usual time and by resolution improvements achieved in the same amount of time.

Non-linear signal detection improvement by radiation damping in single-pulse NMR spectra.

When NMR lines overlap and at least one of them is affected by radiation damping, the resonance line shapes of all lines are no longer Lorentzian. We report the appearance of narrow signal distortions, which resemble hole-burnt spectra. This new experimental phenomenon facilitates the detection of tiny signals hidden below the main resonance. Theoretical analysis based on modified Maxwell-Bloch equations shows that the presence of strong transverse magnetization creates a feedback through the coil, which influences the magnetization of all spins with overlapping resonance lines. In the time domain this leads to cross-precession terms between magnetization densities, which ultimately cause non-linear behavior. Numerical simulations corroborate this interpretation.

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5' position, which allows 5'-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

Determination of the xanthate group distribution on viscose by liquid-state 1H NMR spectroscopy.

An analytical method for determination of the xanthate group distribution on viscoses based on liquid-state NMR spectroscopy was developed. Sample preparation involves stabilization of the xanthate group by allylation followed by derivatization of the remaining free hydroxyl groups at the glucose unit. The method was applied for studying (1) the γ-value (number of xanthate groups per 100 glucose units) of viscose, (2) the distribution of the xanthate groups on the anhydroglucose unit (AGU), and (3) changes of the xanthate group distribution during ripening. Results of the γ-value determination are well comparable with reference methods. Elucidation of the xanthate group distribution on the AGU gives the percentage at the C-6 position and a cumulative share of the positions C-2 and C-3. During ripening, xanthate groups at C-2 and C-3 degrade first, while xanthates at C-6 decompose at a slower rate.

Determination of (3)J((1)H3'-(31)P) couplings in a DNA oligomer with enhanced sensitivity employing a constant-time TOCSY difference experiment.

A constant-time TOCSY difference experiment for the determination of (3)J((1)H3'-(31)P) coupling constants in non-isotope-labelled DNA oligonucleotides is presented. The method is tested on a DNA octamer and compared with the established constant-time NOESY difference method. Each (3)J((1)H3'-(31)P) coupling constant is determined from amplitude changes caused by phosphorous decoupling, which are observable on multiple cross-peaks, thus leading to a high accuracy of the value of the (3)J((1)H3'-(31)P) coupling constant. The new experiment delivers up to three times the sensitivity compared with previously reported methods.

Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA.

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing. The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1ρ experiments with both isotopically labeled and unlabeled RNA samples.

Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy.

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R1ρ relaxation dispersion NMR spectroscopy (RD) determines chemical shift differences between the most stable, ground state and the short-lived, low-populated excited states. We describe a procedure for deducing the excited state structure from these chemical shift differences using the mutate-and-chemical-shift-fingerprint (MCSF) method, which requires ~2-6 weeks and moderate understanding of NMR and RNA structure. We recently applied the MCSF methodology to elucidate the excited state of microRNA 34a targeting the SIRT1 mRNA and use this example to demonstrate the analysis. The protocol comprises the following steps: (i) determination of the secondary structure of the excited state from RD chemical shift data, (ii) design of trapped excited state RNA, (iii) validation of the excited state structure by NMR, and (iv) MCSF analysis comparing the chemical shifts of the trapped excited state with the RD-derived chemical shift differences. MCSF enables observation of the short-lived RNA structures, which can be functionally and structurally characterized by entrapment.

1H, 13C and 15N chemical shift assignment of the stem-loop 5a from the 5'-UTR of SARS-CoV-2.

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.

Signal enhancement in protein NMR using the spin-noise tuning optimum.

We have assessed the potential of an alternative probe tuning strategy based on the spin-noise response for application in common high-resolution multi-dimensional biomolecular NMR experiments with water signal suppression on aqueous and salty samples. The method requires the adjustment of the optimal tuning condition, which may be offset by several 100 kHz from the conventional tuning settings using the noise response of the water protons as an indicator. Although the radio frequency-pulse durations are typically longer under such conditions, signal-to-noise gains of up to 22% were achieved. At salt concentrations up to 100 mM a substantial sensitivity gain was observed.