Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

High-Resolution Structure of RNA G-Quadruplex Containing Unique Structural Motifs Originating from the 5'-UTR of Human Tyrosine Kinase 2 (TYK2).

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor-associated tyrosine kinases together with highly homologous JAK1, JAK2, and JAK3 paralogues. Overexpression of TYK2 is associated with several inflammatory diseases, including severe complications during the COVID-19 infection. Since the downregulation of JAK paralogues could lead to serious health consequences or even death, it is critical to avoid it when designing drugs to suppress TYK2. To achieve the required specificity only for TYK2, researchers have recently selectively targeted TYK2 mRNA by developing antisense oligonucleotides. In this work, we expand the target space of TYK2 mRNA by showing that the mRNA adopts tetra-helical noncanonical structures called G-quadruplexes. We identified a TYKwt RNA oligonucleotide from the 5'-UTR of TYK2 mRNA, which adopts multiple different parallel G-quadruplexes that exist at equilibrium. Using NMR spectroscopy, we showed that some of the G-quadruplexes adopt unique structural motifs, mainly due to the formation of a stable GA bulge. Using guanine to uridine substitutions, we prepared the oligonucleotide TYK3_U6, which serves as an excellent model for the bulged G-quadruplexes formed by the TYKwt oligonucleotide. NMR structural analysis, including data on the residual coupling constants (RDC) of the loop regions, unveiled that the studied three-quartet parallel G-quadruplex contains many unusual structural features such as a G(U)A bulge, a guanine residue in the syn conformation, A and U residues stacked on the top G-quartet, and a well-defined adenine from a three-residue long propeller loop oriented in the groove, all of which could be valuable targets for future drug design.

Structure of native four-repeat satellite III sequence with non-canonical base interactions.

Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5'-ATGGA ATGGA ATGGA ATGGA-3' using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05-0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G-G intercalation sandwiched by sheared A-G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A-T/T-A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

Dimeric structures of DNA ATTTC repeats promoted by divalent cations.

Structural studies of repetitive DNA sequences may provide insights why and how certain repeat instabilities in their number and nucleotide sequence are managed or even required for normal cell physiology, while genomic variability associated with repeat expansions may also be disease-causing. The pentanucleotide ATTTC repeats occur in hundreds of genes important for various cellular processes, while their insertion and expansion in noncoding regions are associated with neurodegeneration, particularly with subtypes of spinocerebellar ataxia and familial adult myoclonic epilepsy. We describe a new striking domain-swapped DNA-DNA interaction triggered by the addition of divalent cations, including Mg2+ and Ca2+. The results of NMR characterization of d(ATTTC)3 in solution show that the oligonucleotide folds into a novel 3D architecture with two central C:C+ base pairs sandwiched between a couple of T:T base pairs. This structural element, referred to here as the TCCTzip, is characterized by intercalative hydrogen-bonding, while the nucleobase moieties are poorly stacked. The 5'- and 3'-ends of TCCTzip motif are connected by stem-loop segments characterized by A:T base pairs and stacking interactions. Insights embodied in the non-canonical DNA structure are expected to advance our understanding of why only certain pyrimidine-rich DNA repeats appear to be pathogenic, while others can occur in the human genome without any harmful consequences.

In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics.

In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and in vitro crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.

Effects of protein G-quadruplex interactions on phase transitions and protein aggregation.

We show that human protein Znf706 interacts specifically with stable, non-canonical nucleic acid structures known as G-quadruplexes. Znf706, though only 76 residues long, is comprised of two distinct domains, one disordered and one ordered. The disordered domain is homologous to the SERF family of proteins and acts to accelerate amyloid formation. The ordered domain contains a C2H2 type zinc-finger. We show that Znf706 not only accelerates amyloid formation but also accelerates amorphous protein aggregation. We find that Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, whose dynamics are constrained upon interaction. G-quadruplexes are potent anti-aggregation agents, and their binding to Znf706 suppresses Znf706's ability to accelerate protein aggregation and fibril formation. Znf706 in conjunction with G-quadruplexes thus may play a role in regulating protein folding. Depletion of Znf706 specifically impacts mRNA abundance of genes that contain high G-quadruplex density, implying that Znf706 may also serve as a G-quadruplex specific regulator. Our studies give insights into how proteins and G-quadruplexes interact, how these interactions affect both partners, lead to liquid-liquid phase transitions, and lead to the modulation of protein aggregation and cellular mRNA levels.

Insight into Tetramolecular DNA G-Quadruplexes Associated with ALS and FTLD: Cation Interactions and Formation of Higher-Ordered Structure.

The G4C2 hexanucleotide repeat expansion in the c9orf72 gene is a major genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), with the formation of G-quadruplexes directly linked to the development of these diseases. Cations play a crucial role in the formation and structure of G-quadruplexes. In this study, we investigated the impact of biologically relevant potassium ions on G-quadruplex structures and utilized 15N-labeled ammonium cations as a substitute for K+ ions to gain further insights into cation binding and exchange dynamics. Through nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we demonstrate that the single d(G4C2) repeat, in the presence of 15NH4+ ions, adopts a tetramolecular G-quadruplex with an all-syn quartet at the 5'-end. The movement of 15NH4+ ions through the central channel of the G-quadruplex, as well as to the bulk solution, is governed by the vacant cation binding site, in addition to the all-syn quartet at the 5'-end. Furthermore, the addition of K+ ions to G-quadruplexes folded in the presence of 15NH4+ ions induces stacking of G-quadruplexes via their 5'-end G-quartets, leading to the formation of stable higher-ordered species.

A Screening Protocol for Exploring Loop Length Requirements for the Formation of a Three Cytosine-Cytosine+ Base-Paired i-Motif.

DNA sequences containing at least four runs of repetitive cytosines can fold into tetra-helical structures called i-Motifs (iMs). The interest in these DNA secondary structures is increasing due to their therapeutical and technological applications. Still, limited knowledge of their folding requirements is currently available. We developed a novel step-by-step pipeline for the systematic screening of putative iM-forming model sequences. Focusing on structures comprising only three cytosine-cytosine+ base pairs, we investigated what the minimal lengths of the loops required for formation of an intra-molecular iM are. Our data indicate that two and three nucleotides are required to connect the strands through the minor and majorgrooves of the iM, respectively. Additionally, they highlight an asymmetric behavior according to the distribution of the cytosines. Specifically, no sequence containing a single cytosine in the first and third run was able to fold into intra-molecular iMs with the same stability of those formed when the first and the third run comprise two cytosines. This knowledge represents a step forward toward the development of prediction tools for the proper identification of biologically functional iMs, as well as for the rational design of these secondary structures as technological devices.

Unveiling the structural mechanism of a G-quadruplex pH-Driven switch.

The human telomere oligonucleotide, d[TAGGG(TTAGGG)2TTAGG] (TAGGG), can adopt two distinct 2-G-quartet G-quadruplex structures at pH 7.0 and 5.0, referred to as the TD and KDH+ forms, respectively. By using a combination of NMR and computational techniques, we determined high-resolution structures of both forms, which revealed unique loop architectures, base triples, and base pairs that play a crucial role in the pH-driven structural transformation of TAGGG. Our study demonstrated that TAGGG represents a reversible pH-driven switch system where the stability and pH-induced structural transformation of the G-quadruplexes are influenced by the terminal residues and base triples. Gaining insight into the factors that regulate the formation of G-quadruplexes and their pH-sensitive structural equilibrium holds great potential for the rational design of novel DNA based pH-driven switches. These advancements in understanding create exciting opportunities for applications in the field of nanotechnology, specifically in the development of bio-nano-motors.

Cytotoxicity and Antibacterial Efficacy of Betaine- and Choline-Substituted Polymers.

Cationic charge has been widely used to increase polymer adsorption and flocculation of dispersions or to provide antimicrobial activity. In this work, cationization of hydroxyethyl cellulose (HEC) and polyvinyl alcohol (PVA) was achieved by covalently coupling betaine hydrochloride and choline chloride to the polymer backbones through carbonyl diimidazole (CDI) activation. Two approaches for activation were investigated. CDI in excess was used to activate the polymers' hydroxyls followed by carbonate formation with choline chloride, or CDI was used to activate betaine hydrochloride, followed by ester formation with the polymers' hydroxyls. The first approach led to a more significant cross-linking of PVA, but not of HEC, and the second approach successfully formed ester bonds. Cationic, nitrogen-bearing materials with varying degrees of substitution were obtained in moderate to high yields. These materials were analyzed by Fourier transform infrared spectroscopy, nuclear magnetic resonance, polyelectrolyte titration, and kaolin flocculation. Their dose-dependent effect on the growth of Staphylococcus aureus and Pseudomonas aeruginosa, and L929 mouse fibroblasts, was investigated. Significant differences were found between the choline- and betaine-containing polymers, and especially, the choline carbonate esters of HEC strongly inhibited the growth of S. aureus in vitro but were also cytotoxic to fibroblasts. Fibroblast cytotoxicity was also observed for betaine esters of PVA but not for those of HEC. The materials could potentially be used as antimicrobial agents for instance by coating surfaces, but more investigations into the interaction between cells and polysaccharides are necessary to clarify why and how bacterial and human cells are inhibited or killed by these derivatives, especially those containing choline.

Spatial arrangement of functional domains in OxyS stress response sRNA.

Small noncoding RNAs are an important class of regulatory RNAs in bacteria, often regulating responses to changes in environmental conditions. OxyS is a 110 nt, stable, trans-encoded small RNA found in Escherichia coli and is induced by an increased concentration of hydrogen peroxide. OxyS has an important regulatory role in cell stress response, affecting the expression of multiple genes. In this work, we investigated the structure of OxyS and the interaction with fhlA mRNA using nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and unbiased molecular dynamics simulations. We determined the secondary structures of isolated stem-loops and confirmed their structural integrity in OxyS. Unexpectedly, stem-loop SL4 was identified in the region that was predicted to be unstructured. Three-dimensional models of OxyS demonstrate that OxyS adopts an extended structure with four solvent-exposed stem-loops, which are available for interaction with other RNAs and proteins. Furthermore, we provide evidence of base-pairing between OxyS and fhlA mRNA.

Thiocarbohydrazone and Chalcone-Derived 3,4-Dihydropyrimidinethione as Lipid Peroxidation and Soybean Lipoxygenase Inhibitors.

The potential of the 4,6-diphenyl-3,4-dihydropyrimidine-2(1H)-thione (abbreviated as KKII5) and (E)-N'-benzylidenehydrazinecarbothiohydrazide (abbreviated as DKI5) compounds as possible drug leads is investigated. KKII5 and DKI5 are synthesized in high yield of up to 97%. Their structure, binding in the active site of the LOX-1 enzyme, and their toxicity are studied via joint experimental and computational methodologies. Specifically, the structure assignment and conformational analysis were achieved by applying homonuclear and heteronuclear 2D nuclear magnetic resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-HSQC, and 2D-HMBC) and density functional theory (DFT). The obtained DFT lowest energy conformers were in agreement with the NOE correlations observed in the 2D-NOESY spectra. Additionally, docking and molecular dynamics simulations were performed to discover their ability to bind and remain stabile in the active site of the LOX-1 enzyme. These in silico experiments and DFT calculations indicated favorable binding for the enzyme under study. The strongest binding energy, -9.60 kcal/mol, was observed for dihydropyrimidinethione KKII5 in the active site of LOX-1. ADMET calculations showed that the two molecules lack major toxicities and could serve as possible drug leads. The redox potential of the active center of LOX-1 with the binding molecules was calculated via DFT methodology. The results showed a significantly smaller energy attachment of 2.8 eV with KKII5 binding in comparison to DKI5. Thus, KKII5 enhanced the ability of the active center to receive electrons compared to DKI5. This is related to the stronger binding interaction of KKII5 relative to that of DK15 to LOX-1. The two very potent LOX-1 inhibitors exerted IC50 19 µΜ (KKII5) and 22.5 µΜ (DKI5). Furthermore, they both strongly inhibit lipid peroxidation, namely, 98% for KKII5 and 94% for DKI5.

Efficiency of Neat and Quaternized-Cellulose Nanofibril Fillers in Chitosan Membranes for Direct Ethanol Fuel Cells.

In this work, fully polysaccharide based membranes were presented as self-standing, solid polyelectrolytes for application in anion exchange membrane fuel cells (AEMFCs). For this purpose, cellulose nanofibrils (CNFs) were modified successfully with an organosilane reagent, resulting in quaternized CNFs (CNF (D)), as shown by Fourier Transform Infrared Spectroscopy (FTIR), Carbon-13 (C13) nuclear magnetic resonance (13C NMR), Thermogravimetric Analysis (TGA)/Differential Scanning Calorimetry (DSC), and ζ-potential measurements. Both the neat (CNF) and CNF(D) particles were incorporated in situ into the chitosan (CS) membrane during the solvent casting process, resulting in composite membranes that were studied extensively for morphology, potassium hydroxide (KOH) uptake and swelling ratio, ethanol (EtOH) permeability, mechanical properties, ionic conductivity, and cell performance. The results showed higher Young's modulus (119%), tensile strength (91%), ion exchange capacity (177%), and ionic conductivity (33%) of the CS-based membranes compared to the commercial Fumatech membrane. The addition of CNF filler improved the thermal stability of the CS membranes and reduced the overall mass loss. The CNF (D) filler provided the lowest (4.23 × 10-5 cm2 s-1) EtOH permeability of the respective membrane, which is in the same range as that of the commercial membrane (3.47 × 10-5 cm2s-1). The most significant improvement (~78%) in power density at 80 °C was observed for the CS membrane with neat CNF compared to the commercial Fumatech membrane (62.4 mW cm-2 vs. 35.1 mW cm-2). Fuel cell tests showed that all CS-based anion exchange membranes (AEMs) exhibited higher maximum power densities than the commercial AEMs at 25 °C and 60 °C with humidified or non-humidified oxygen, demonstrating their potential for low-temperature direct ethanol fuel cell (DEFC) applications.

Structural polymorphism driven by a register shift in a CGAG-rich region found in the promoter of the neurodevelopmental regulator AUTS2 gene.

The AUTS2 gene has been shown to influence brain development by controlling the number of neurons, promoting the growth of axons and dendrites and regulating neuronal migration. The expression of two isoforms of AUTS2 protein is precisely regulated and misregulation of their expression has been correlated with neurodevelopmental delay and autism spectrum disorder. A CGAG-rich region, which includes a putative protein binding site (PPBS), d(AGCGAAAGCACGAA), was found in the promoter region of AUTS2 gene. We show that oligonucleotides from this region adopt thermally stable non-canonical hairpin structures stabilized by G:C and sheared G:A base pairs arranged in a repeating structural motif we termed CGAG block. These motifs are formed consecutively, in a way that exploits a shift in register throughout the whole CGAG repeat to maximize the number of consecutive G:C and G:A base pairs. The differences in CGAG repeat shifting affect the structure of the loop region, where PPBS residues are predominantly located, specifically the loop length, types of base pairs and the pattern of base-base stacking. Finally, we propose a previously unexplored mechanism, by which different folds in the CGAG-rich region could cause a switch in expression between the full-length and C-terminal isoforms of AUTS2.

NMR of RNA - Structure and interactions.

RNA was shown to have a more substantial role in the regulation of diverse cellular processes than anticipated until recently. Answers to questions what is the structure of specific RNAs, how structure changes to accommodate different functional roles, and how RNA senses other biomolecules and changes its fold upon interaction create a complete representation of RNA involved in cellular processes. Nuclear magnetic resonance (NMR) spectroscopy encompasses a collection of methods and approaches that offer insight into several structural aspects of RNAs. We review the most recent advances in the field of viral, long non-coding, regulatory, and four-stranded RNAs, with an emphasis on the detection of dynamic sub-states and in view of chemical modifications that expand RNA's function.

Identification and characterisation of G-quadruplex DNA-forming sequences in the Pseudomonas aeruginosa genome.

A number of Gram-negative bacteria such as Pseudomonas aeruginosa are becoming resistant to front-line antibiotics. Consequently, there is a pressing need to find alternative bio-molecular targets for the development of new drugs. Since non-canonical DNA structures such as guanine-quadruplexes (G4s) have been implicated in regulating transcription, we were interested in determining whether there are putative quadruplex-forming sequences (PQS) in the genome of Pseudomonas aeruginosa. Using bioinformatic tools, we screened 36 genes potentially relevant to drug resistance for the presence of PQS and 10 of these were selected for biophysical characterisation (i.e. circular dichroism and thermal difference UV/Vis spectroscopy). These studies showed that three of these G-rich sequences (linked to murE, ftsB and mexC genes) form stable guanine-quadruplexes which were studied by NMR spectroscopy; detailed analysis of one of the sequences (mexC) confirmed that it adopts a two-quartet antiparallel quadruplex structure in the presence of K+ ions. We also show by FRET melting assays that small molecules can stabilise these three new G4 DNA structures under physiological conditions. These initial results could be of future interest in the development of new antibiotics with alternative bio-molecular targets which in turn would help tackle antimicrobial resistance.

G-Quadruplex Binding of an NIR Emitting Osmium Polypyridyl Probe Revealed by Solution NMR and Time-Resolved Infrared Studies.

G-quadruplexes are emerging targets in cancer research and understanding how diagnostic probes bind to DNA G-quadruplexes in solution is critical to the development of new molecular tools. In this study the binding of an enantiopure NIR emitting [Os(TAP)2 (dppz)]2+ complex to different G-quadruplex structures formed by human telomer (hTel) and cMYC sequences in solution is reported. The combination of NMR and time-resolved infrared spectroscopic techniques reveals the sensitivity of the emission response to subtle changes in the binding environment of the complex. Similar behaviour is also observed for the related complex [Os(TAP)2 (dppp2)]2+ upon quadruplex binding.

Children gut microbiota exhibits a different composition and metabolic profile after in vitro exposure to Clostridioides difficile and increases its sporulation.

Clostridioides difficile (Clostridium difficile) infection (CDI) is one of the main public health concerns in adults, while children under 2 years of age are often colonized asymptomatically. In both adults and children, CDI is strongly associated with disturbances in gut microbiota. In this study, an in-vitro model of children gut microbiota was challenged with vegetative cells or a conditioned media of six different toxigenic C. difficile strains belonging to the ribotypes 027, 078, and 176. In the presence of C. difficile or conditioned medium the children gut microbiota diversity decreased and all main phyla (Bacteroidetes, Firmicutes, and Proteobacteria) were affected. The NMR metabolic spectra divided C. difficile exposed children gut microbiota into three clusters. The grouping correlated with nine metabolites (short chain fatty acids, ethanol, phenolic acids and tyramine). All strains were able to grow in the presence of children gut microbiota and showed a high sporulation rate of up to 57%. This high sporulation rate in combination with high asymptomatic carriage in children could contribute to the understanding of the reported role of children in C. difficile transmissions.

8-Oxoguanine Forms Quartets with a Large Central Cavity.

Oxidation of a guanine nucleotide in DNA yields an 8-oxoguanine nucleotide (oxoG) and is a mutagenic event in the genome. Due to different arrangements of hydrogen-bond donors and acceptors, oxoG can affect the secondary structure of nucleic acids. We have investigated base pairing preferences of oxoG in the core of a tetrahelical G-quadruplex structure, adopted by analogues of d(TG4T). Using spectroscopic methods, we have shown that G-quartets can be fully substituted with oxoG nucleobases to form an oxoG-quartet with a revamped hydrogen-bonding scheme. While an oxoG-quartet can be incorporated into the G-quadruplex core without distorting the phosphodiester backbone, larger dimensions of the central cavity change the cation localization and exchange properties.