Solution-State Structure of a Long-Loop G-Quadruplex Formed Within Promoters of Plasmodium falciparum B var Genes.

We report the high-resolution NMR solution-state structure of an intramolecular G-quadruplex with a diagonal loop of ten nucleotides. The G-quadruplex is formed by a 34-nt DNA sequence, d[CAG3T2A2G3TATA2CT3AG4T2AG3T2], named UpsB-Q-1. This sequence is found within promoters of the var genes of Plasmodium falciparum, which play a key role in malaria pathogenesis and evasion of the immune system. The [3+1]-hybrid G-quadruplex formed under physiologically relevant conditions exhibits a unique equilibrium between two structures, both stabilized by base stacking and non-canonical hydrogen bonding. Unique equilibrium of the two closely related 3D structures originates from a North-South repuckering of deoxyribose moiety of residue T27 in the lateral loop. Besides the 12 guanines involved in three G-quartets, most residues in loop regions are involved in interactions at both G-quartet-loop interfaces.

miRNA and DNA analysis by negative ion electron transfer dissociation and infrared multiple-photon dissociation mass spectrometry.

BACKGROUND: The use of simple and hybrid fragmentation techniques for the identification of molecules in tandem mass spectrometry provides different and complementary information on the structure of molecules. Nevertheless, these techniques have not been as widely explored for oligonucleotides as for peptides or proteins. The analysis of microRNAs (miRNAs) warrants special attention, given their regulatory role and their relationship with several diseases. The application of different fragmentation techniques will be very interesting for their identification. RESULTS: Four synthetic miRNAs and a DNA sequence were fragmented in an ESI-FT-ICR mass spectrometer using both simple and hybrid fragmentation techniques: CID, nETD followed by CID, IRMPD, and, for the first time, nETD in combination with IRMPD. The main fragmentation channel was base loss. The use of nETD-IRMPD resulted in d/z, a/w, and c/y ions at higher intensities. Moreover, nETD-IRMPD provided high sequence coverage and low internal fragmentation. Native MS analysis revealed that only miR159 and the DNA sequence formed stable dimers under physiological ionic strength. The use of organic co-solvents or additives resulted in a lower sequence coverage due to lesser overall ionization efficiency. NOVELTY: This work demonstrates that the combination of nETD and IRMPD for miRNA fragmentation constitutes a suitable alternative to common fragmentation methods. This strategy resulted in efficient fragmentation of [miRNA]5- using low irradiation times and fewer internal fragments while ensuring a high sequence coverage. Moreover, given that such low charge states predominate upon spraying in physiological-like conditions, native MS can be applied for obtaining structural information at the same time.

Mutations in Tau Protein Promote Aggregation by Favoring Extended Conformations.

Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Some tauopathies can be inherited due to mutations in the gene encoding tau, which might favor the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the disease-associated single-point mutations promote amyloid formation. We combined biochemical and biophysical characterization, notably, small-angle X-ray scattering (SAXS), to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions, and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radii of gyration. We show that mutations disfavor intramolecular protein interactions, which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate the aggregation propensity of IDPs.

Molecular Insights into O-Linked Sialoglycans Recognition by the Siglec-Like SLBR-N (SLBRUB10712) of Streptococcus gordonii.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

New 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline Derivatives: Synthesis and Biological Evaluation as Novel Anticancer Agents by Targeting G-Quadruplex.

The syntheses of novel 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12 and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinolines 13 are reported here in six steps starting from various halogeno-quinazoline-2,4-(1H,3H)-diones or substituted anilines. The antiproliferative activities of the products were determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. Disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines 12b, 12f, and 12i displayed the most interesting antiproliferative activities against six human cancer cell lines. In the series of quinoline derivatives, 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline 13a proved to be the most active. G-quadruplexes (G4) stacked non-canonical nucleic acid structures found in specific G-rich DNA, or RNA sequences in the human genome are considered as potential targets for the development of anticancer agents. Then, as small aza-organic heterocyclic derivatives are well known to target and stabilize G4 structures, their ability to bind G4 structures have been determined through FRET melting, circular dichroism, and native mass spectrometry assays. Finally, telomerase inhibition ability has been also assessed using the MCF-7 cell line.