Nuclear Magnetic Resonance reveals a two hairpin equilibrium near the 3'-splice site of Influenza A segment 7 mRNA that can be shifted by oligonucleotides

Influenza A kills hundreds of thousands of people globally every year and has potential to generate more severe pandemics. Influenza A’s RNA genome and transcriptome provide many potential therapeutic targets. Here, nuclear magnetic resonance (NMR) experiments suggest that one such target could be a hairpin loop of eight nucleotides in a pseudoknot that sequesters a 3' splice site in canonical pairs until a conformational change releases it into a dynamic 2X2 nucleotide internal loop. NMR experiments reveal that the hairpin loop is dynamic and able to bind oligonucleotides as short as pentamers. A 3D NMR structure of the complex contains four and likely five base pairs between pentamer and loop. Moreover, a hairpin sequence was discovered that mimics the equilibrium of the influenza hairpin between its structure in the pseudoknot and upon release of the splice site. Oligonucleotide binding shifts the equilibrium completely to the hairpin secondary structure required for pseudoknot folding. The results suggest this hairpin can be used to screen for compounds that stabilize the pseudoknot and potentially reduce splicing.

Quick-Look Water Saturation Estimate with Density-Neutron Logs in Unknown or Mixed Salinity Environments: Case Studies in Middle East Oil-Bearing Carbonate Reservoirs

We have shown previously that while total porosity is the weighted sum of density and neutron porosities, hydrocarbon volume is the weighted difference of the two. Thus, their ratio yields hydrocarbon, or equivalently, water saturation (Sw). In LWD environments where negligible invasion takes place while drilling, we investigate whether Sw derived from LWD density-neutron logs could approach true Sw in unknown or mixed water salinity environments. In such environments, it is well known that Sw determined from standalone resistivity or capture sigma logs is uncertain due to large water resistivity (Rw) or capture sigma (Σw) changes with salinity. On the other hand, the water density (ρw) and hydrogen index (HIw) variations with salinity are much less (Table 1). Hence, the water point on the density neutron crossplot does not move with salinity as much as the water point on a sigma-porosity crossplot does. Similarly, the water point on a resistivity-porosity Pickett plot would move drastically with changes in Rw. Also, because the hydrocarbon effect on density-neutron logs is much less in oil than in gas, the weights in the density-neutron porosities can be conveniently set at midpoint in light oil-bearing reservoirs without compromising porosity and saturation results. Thus, a quicklook estimate of Sw from density-neutron logs is the normalized ratio of the difference over the sum of density and neutron porosities. The normalization factor is a function of the hydrocarbon density. We also build a graphical Sw overlay for petrophysical insights. We tested the LWD density-neutron derived Sw in two Middle East carbonate oil wells that have mixed salinity. The two wells were extensively studied in the past. In the first well, the reference Sw is given by the joint-inversion of resistivity-sigma logs, corroborated with Sw estimated from multi-measurements time-lapsed analysis, and validated with water analysis on water samples taken by formation testers. In the second well, comprehensive wireline measurements targeting mixed salinity such as dielectric and 3D NMR were acquired to derive Sw, and complemented by formation tester sampling, core measurements, and LWD resistivity-sigma Sw. In both wells, density-neutron quicklook Sw agrees surprisingly well with Sw from other techniques. It may lack the accuracy and precision and the continuous salinity output but is sufficient to pinpoint both flooded zones and bypassed oil zones. Since density-neutron is part of triple-combo data that are first available in well data acquisition, it is recommended to go beyond porosity application and compute water saturation (Sw) in unknown or mixed salinity environments. The computation is straightforward and can be useful to complement other established techniques for quick evaluation in unknown or mixed water salinity environments.

A functional SNP regulates E-cadherin expression by dynamically remodeling the 3D structure of a promoter-associated non-coding RNA transcript

Transcription of E-cadherin, a tumor suppressor which plays critical roles in cell adhesion and the epithelial-mesenchymal transition, is regulated by a promoter-associated non-coding transcript. This RNA includes a functional C/A single nucleotide polymorphism (SNP rs16260). The A-allele is linked to decreased transcriptional activity and increased prostate cancer risk. This single nucleotide change affects recruitment of an iso-miRNA and epigenetic enzymes to regulate the promoter, yet it is distant from the isomiR-binding site in both primary sequence and secondary structure, raising the question of how regulation occurs. Here we report the 3D NMR structure of the domain of 90 nucleotides within the paRNA which includes the SNP and the isomiR-binding site. We show that the A->C mutation alters the locally dynamic structure of the paRNA, revealing that the mutation regulates the E-cadherin promoter through its effect on RNA structure.

Introduction to a special issue of <i>Magnetic Resonance</i> in honour of Robert Kaptein at the occasion of his 80th birthday

This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from colleagues, many of whom have worked with him, and others who admire his work and have been stimulated by his research. The contributions show current research in biomolecular NMR, spin hyperpolarisation and spin chemistry, including CIDNP (chemically induced dynamic nuclear polarisation), topics to which he has contributed enormously. His proposal of the radical pair mechanism was the birth of the field of spin chemistry, and the laser CIDNP NMR experiment on a protein was a major breakthrough in hyperpolarisation research. He set milestones for biomolecular NMR by developing computational methods for protein structure determination, including restrained molecular dynamics and 3D NMR methodology. With a lac repressor headpiece, he determined one of the first protein structures determined by NMR. His studies of the lac repressor provided the first examples of detailed studies of protein nucleic acid complexes by NMR. This deepened our understanding of protein DNA recognition and led to a molecular model for protein sliding along the DNA. Furthermore, he played a leading role in establishing the cluster of NMR large-scale facilities in Europe. This editorial gives an introduction to the publication and is followed by a biography describing his contributions to magnetic resonance.

Structure of Nanobody Nb23

Background: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. Results: The solution structure of isolated Nb23 nanobody was determined. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

Determination of protein–ligand binding modes using fast multi-dimensional NMR with hyperpolarization

The structure of a ligand bound to a protein is determined from fast pseudo-3D NMR spectroscopy with transfer of hyperpolarization.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.