The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized to date involve long-lived species that can be captured by structure characterization techniques. Here, we report the Nuclear Magnetic Resonance visualization of RNA transitions towards invisible ‘excited states’ (ES), which exist in too little abundance (2–13%) and for too short periods of time (45–250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix-junction-helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signaling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.

Rare tautomeric and anionic nucleobases are believed to play fundamental biological roles but their prevalence and functional importance has remained elusive because they exist transiently, in low-abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10−3-10−5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

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.

Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle–host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.

Soluble Klotho and 1,25D levels decrease and FGF23 levels increase at early CKD stages, whereas PTH levels increase at more advanced CKD stages.

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2 protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.Kidney International advance online publication,

Determination of the 3D structure of small-to mediumsized organic and biomolecular compounds relies on the use of several NMR parameters. Information about dihedral angles from homo-and heteronuclear 3 J scalar couplings [1][2][3] is usually combined with information on distances from the nuclear Overhauser effect (NOE).[4] Even when using the recently (re)introduced (residual) dipolar couplings [5] to study flexible compounds, [6][7][8] distance information from the NOE is still essential for structure determination. The buildup rate of the NOE, however, depends on the correlation time t c of the compound and the observation frequency w. The NOE changes sign at wt c % 1.12, which leads to the well-known phenomenon that little or no NOE is observed for medium-sized compounds (MW % 1000 g mol À1). This impedes the extraction of distance information from NOESY spectra of these compounds.This problem can be solved by using rotating-frame nuclear Overhauser effect spectroscopy (ROESY), [9,10] which yields negative cross-peaks (corresponding to positive Overhauser enhancements) for all values of wt c . In the interpretation of ROESY, however, several experimental problems-namely direct cross-peaks due to J coupling, Hartmann-Hahn matching (leading to TOCSY, total correlation spectroscopy, cross-peaks) and offset dependence [11] -have to be avoided. The offset dependence influences the crosspeak integrals of all spins depending on their offset, whereas the other two phenomena can degrade the line shapes and integrals of ROESY cross-peaks. TOCSY artefacts also impede the use of ROESY for detecting chemical exchange.[12] These problems lead to serious complications, when distances or exchange rates are to be extracted from ROESY spectra.Several ROESY pulse sequences have been proposed for removing these artefacts, [13][14][15] but they are cumbersome to set up, reduce sensitivity too much, or show pronounced offset dependence. Thus a robust, convenient procedure is needed that yields high-quality, high-sensitivity spectra that can be reliably integrated even in the presence of J coupling.We have previously shown that jump-symmetrized ROESY (JS-ROESY) combines negligible offset dependence with close-to-optimal suppression of TOCSY, [14] but the experimental setup is quite laborious as pulses with finetuned power levels (gray in Figure 1 A) are needed. Here we show that by bracketing two off-resonance spin-lock pulses with adiabatic pulses [16] , we obtain high-quality, highsensitivity spectra with no need for a sample-specific setup (Figure 1 B). The resulting spectra can be reliably quantified and TOCSY transfer is as well suppressed as in conventional JS-ROESY. The superior performance of this convenient (gray) is needed to transfer magnetization from the low-to the high-field spinlock (SL). B) Adiabatic ramps at times a-d transfer magnetization to the spinlocks. After EASY ROESY has been set up once, parameters are calculated automatically. Gradient pulses (G z ) suppress unwanted signals. Solvent suppression can easi...

Self-reported gout is common among patients with CKD and lower GFR is strongly associated with gout. Pharmacological management of gout in patients with CKD is suboptimal. Prospective follow-up will show whether gout and hyperuricaemia increase the risk of CKD progression and cardiovascular events in the GCKD study.