Structural Differences at Quadruplex-Duplex Interfaces Enable Ligand-Induced Topological Transitions.

Quadruplex-duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high-affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem-loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair-type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen-DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism.

A pH-Responsive Topological Switch Based on a DNA Quadruplex-Duplex Hybrid.

A guanine-rich oligonucleotide based on a human telomeric sequence but with the first three-nucleotide intervening stretch replaced by a putative 15-nucleotide hairpin-forming sequence shows a pH-dependent folding into different quadruplex-duplex hybrids in a potassium containing buffer. At slightly acidic pH, the quadruplex domain adopts a chair-type conformation. Upon increasing the pH, a transition with a midpoint close to neutral pH to a major and minor (3+1) hybrid topology with either a coaxially stacked or orthogonally oriented duplex stem-loop occurs. NMR-derived high-resolution structures reveal that an adenine protonation is prerequisite for the formation of a non-canonical base quartet, capping the outer G-tetrad at the quadruplex-duplex interface and stabilizing the antiparallel chair conformation in an acidic environment. Being directly associated with interactions at the quadruplex-duplex interface, this unique pH-dependent topological transition is fully reversible. Coupled with a conformation-sensitive optical readout demonstrated as a proof of concept using the fluorescent dye thiazole orange, the present quadruplex-duplex hybrid architecture represents a potentially valuable pH-sensing system responsive in a physiological pH range of 7±1.

Impact of loop length and duplex extensions on the design of hybrid-type G-quadruplexes.

A G-rich core sequence G3-TCA-G3-T1,2-G3-T1,2-G3 can be designed to fold into a parallel or into two different (3+1) hybrid-type G-quadruplexes, among them an elusive topology with one lateral followed by two propeller loops. Favored folds can be rationalized based on the number of intervening thymidines and on additional complementary flanking sequences.

Showcasing Different G-Quadruplex Folds of a G-Rich Sequence: Between Rule-Based Prediction and Butterfly Effect.

In better understanding the interactions of G-quadruplexes in a cellular or noncellular environment, a reliable sequence-based prediction of their three-dimensional fold would be extremely useful, yet is often limited by their remarkable structural diversity. A G-rich sequence related to a promoter sequence of the PDGFR-ß nuclease hypersensitivity element (NHE) comprises a G3-G3-G2-G4-G3 pattern of five G-runs with two to four G residues. Although the predominant formation of three-layered canonical G-quadruplexes with uninterrupted G-columns can be expected, minimal base substitutions in a non-G-tract domain were shown to guide folding into either a basket-type antiparallel quadruplex, a parallel-stranded quadruplex with an interrupted G-column, a quadruplex with a V-shaped loop, or a (3+1) hybrid quadruplex. A 3D NMR structure for each of the different folds was determined. Supported by thermodynamic profiling on additional sequence variants, formed topologies were rationalized by the identification and assessment of specific critical interactions of loop and overhang residues, giving valuable insights into their contribution to favor a particular conformer. The variability of such tertiary interactions, together with only small differences in quadruplex free energies, emphasizes current limits for a reliable sequence-dependent prediction of favored topologies from sequences with multiple irregularly positioned G-tracts.

High-affinity binding at quadruplex-duplex junctions: rather the rule than the exception.

Quadruplex-duplex (Q-D) junctions constitute unique structural motifs in genomic sequences. Through comprehensive calorimetric as well as high-resolution NMR structural studies, Q-D junctions with a hairpin-type snapback loop coaxially stacked onto an outer G-tetrad were identified to be most effective binding sites for various polycyclic quadruplex ligands. The Q-D interface is readily recognized by intercalation of the ligand aromatic core structure between G-tetrad and the neighboring base pair. Based on the thermodynamic and structural data, guidelines for the design of ligands with enhanced selectivity towards a Q-D interface emerge. Whereas intercalation at Q-D junctions mostly outcompete stacking at the quadruplex free outer tetrad or intercalation between duplex base pairs to varying degrees, ligand side chains considerably contribute to the selectivity for a Q-D target over other binding sites. In contrast to common perceptions, an appended side chain that additionally interacts within the duplex minor groove may confer only poor selectivity. Rather, the Q-D selectivity is suggested to benefit from an extension of the side chain towards the exposed part of the G-tetrad at the junction. The presented results will support the design of selective high-affinity binding ligands for targeting Q-D interfaces in medicinal but also technological applications.

Guiding the folding of G-quadruplexes through loop residue interactions.

A G-rich sequence was designed to allow folding into either a stable parallel or hybrid-type topology. With the parent sequence featuring coexisting species, various related sequences with single and double mutations and with a shortened central propeller loop affected the topological equilibrium. Two simple modifications, likewise introduced separately to all sequences, were employed to lock folds into one of the topologies without noticeable structural alterations. The unique combination of sequence mutations, high-resolution NMR structural information, and the thermodynamic stability for both topological competitors identified critical loop residue interactions. In contrast to first loop residues, which are mostly disordered and exposed to solvent in both propeller and lateral loops bridging a narrow groove, the last loop residue in a lateral three-nucleotide loop is engaged in stabilizing stacking interactions. The propensity of single-nucleotide loops to favor all-parallel topologies by enforcing a propeller-like conformation of an additional longer loop is shown to result from their preference in linking two outer tetrads of the same tetrad polarity. Taken together, the present studies contribute to a better structural and thermodynamic understanding of delicate loop interactions in genomic and artificially designed quadruplexes, e.g. when employed as therapeutics or in other biotechnological applications.

The role of FoxO1 and its modulation with small molecules in the development of diabetes mellitus: A review.

Diabetes mellitus type 2 (T2D) is one of the metabolic disorders suffered by a global human being. Certain factors, such as lifestyle and heredity, can increase a person's tendency for T2D. Various genes and proteins play a role in the development of insulin resistance and ultimately diabetes in which one central protein that is discussed in this review is FoxO1. In this review, we regard FoxO1 activation as detrimental, promote high plasma glucose level, and induce insulin resistance. Indeed, many contrasting studies arise since FoxO1 is an important protein to alleviate oxidative stress and promote cell survival, for example, also by preventing hyperglycemic-induced cell death. Inter-relation to PPARG, another important protein in metabolism, is also discussed. Ultimately, we discussed contrasting approaches of targeting FoxO1 to combat diabetes mellitus by small molecules.

Indoloquinoline Ligands Favor Intercalation at Quadruplex-Duplex Interfaces.

Quadruplex-duplex (Q-D) junctions are increasingly considered promising targets for medicinal and technological applications. Here, a Q-D hybrid with a hairpin-type snapback loop coaxially stacked onto the quadruplex 3'-outer tetrad was designed and employed as a target structure for the indoloquinoline ligand SYUIQ-5. NMR spectral analysis demonstrated high-affinity binding of the ligand at the quadruplex-duplex interface with association constants determined by isothermal titration calorimetry of about 107  M-1 and large exothermicities ΔH° of -14 kcal/mol in a 120 mM K+ buffer at 40 °C. Determination of the ligand-bound hybrid structure revealed intercalation of SYUIQ-5 between 3'-outer tetrad and the neighboring CG base pair, maximizing π-π stacking as well as electrostatic interactions with guanine carbonyl groups in close vicinity to the positively charged protonated quinoline nitrogen of the tetracyclic indoloquinoline. Exhibiting considerable flexibility, the SYUIQ-5 sidechain resides in the duplex minor groove. Based on comparative binding studies with the non-substituted N5-methylated indoloquinoline cryptolepine, the sidechain is suggested to confer additional affinity and to fix the alignment of the intercalated indoloquinoline aromatic core. However, selectivity for the Q-D junction mostly relies on the geometry and charge distribution of the indoloquinoline ring system. The presented results are expected to provide valuable guidelines for the design of ligands specifically targeting Q-D interfaces.

Structural motifs and intramolecular interactions in non-canonical G-quadruplexes.

Guanine(G)-rich DNA or RNA sequences can assemble or intramolecularly fold into G-quadruplexes formed through the stacking of planar G·G·G·G tetrads in the presence of monovalent cations. These secondary nucleic acid structures have convincingly been shown to also exist within a cellular environment exerting important regulatory functions in physiological processes. For identifying nucleic acid segments prone to quadruplex formation, a putative quadruplex sequence motif encompassing closely spaced tracts of three or more guanosines is frequently employed for bioinformatic search algorithms. Depending on the number and type of intervening residues as well as on solution conditions, such sequences may fold into various canonical G4 topologies with continuous G-columns. On the other hand, a growing number of sequences capable of quadruplex formation feature G-deficient guanine tracts, escaping the conservative consensus motif. By folding into non-canonical quadruplex structures, they adopt unique topologies depending on their specific sequence context. These include G-columns with only two guanines, bulges, snapback loops, D- and V-shaped loops as well as interlocked structures. This review focuses on G-quadruplex species carrying such distinct structural motifs. It evaluates characteristic features of their non-conventional scaffold and highlights principles of stabilizing interactions that also allow for their folding into stable G-quadruplex structures.

Expanding the Topological Landscape by a G-Column Flip of a Parallel G-Quadruplex.

Canonical G-quadruplexes can adopt a variety of different topologies depending on the arrangement of propeller, lateral, or diagonal loops connecting the four G-columns. A novel intramolecular G-quadruplex structure is derived through inversion of the last G-tract of a three-layered parallel fold, associated with the transition of a single propeller into a lateral loop. The resulting (3+1) hybrid fold features three syn⋅anti⋅anti⋅anti G-tetrads with a 3'-terminal all-syn G-column. Although the ability of forming a duplex stem-loop between G-tracts seems beneficial for a propeller-to-lateral loop rearrangement, unmodified G-rich sequences resist folding into the new (3+1) topology. However, refolding can be driven by the incorporation of syn-favoring guanosine analogues into positions of the fourth G-stretch. The presented hybrid-type G-quadruplex structure as determined by NMR spectroscopy may provide for an additional scaffold in quadruplex-based technologies.

First Tandem Repeat of a Potassium Channel KCNN4 Minisatellite Folds into a V-Loop G-Quadruplex Structure.

The KCNN4 gene encoding a potassium channel protein whose expression has been correlated with tumor progression was found to comprise a guanine-rich minisatellite region with the ability to form a putative G-quadruplex (G4). Given the suggested regulatory role of G4s in gene expression, G-quadruplex formation for the polymorphic first repeat of the minisatellite was studied by nuclear magnetic resonance spectroscopy. A stable G-quadruplex of a truncated mutant sequence was shown to represent one of several coexisting species of the wild-type sequence. The high-resolution structure features a noncanonical G4 with a broken G-column and a V-shaped loop. The presence of a 3'-flanking thymidine interacting with the lateral loop preceding the V loop seems to be critical for the formation of this G4 topology. On the contrary, an additional 5'-flanking residue disfavored but still allowed folding into the V-loop structure. The latter may therefore serve as a putative therapeutic target in strategies for G4-based modulation of KCNN4 expression.

G-Quadruplex Formation in a Putative Coding Region of White Spot Syndrome Virus: Structural and Thermodynamic Aspects.

White spot disease (WSD) is one of the most devastating viral infections of crustaceans caused by the white spot syndrome virus (WSSV). A conserved sequence WSSV131 in the DNA genome of WSSV was found to fold into a polymorphic G-quadruplex structure. Supported by two mutant sequences with single G→T substitutions in the third G4 tract of WSSV131, circular dichroism and NMR spectroscopic analyses demonstrate folding of the wild-type sequence into a three-tetrad parallel topology comprising three propeller loops with a major 1 : 3 : 1 and a minor 1 : 2 : 2 loop length arrangement. A thermodynamic analysis of quadruplex formation by differential scanning calorimetry (DSC) indicates a thermodynamically more stable 1 : 3 : 1 loop isomer. DSC also revealed the formation of additional highly stable multimeric species with populations depending on potassium ion concentration.

Quadruplex-Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands.

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5'- or 3'-terminus to mimic a quadruplex-duplex (Q-D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q-D hybrids with a very high affinity in the order Ka ≈107  m-1 irrespective of the duplex location at the quadruplex 3'- or 5'-end. NMR chemical shift perturbations identified the tetrad face of the Q-D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3'- or 5'-terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3'-Q-D junction, whereas non-hydrophobic entropic contributions favor binding with formation of a 2:1 ligand-quadruplex complex in case of the 5'-Q-D hybrid.

The genetic basis of high-carbohydrate and high-monosodium glutamate diet related to the increase of likelihood of type 2 diabetes mellitus: a review.

Diabetes is one of the most common metabolic diseases. Aside from the genetic factor, previous studies stated that other factors such as environment, lifestyle, and paternal-maternal condition play critical roles in diabetes through DNA methylation in specific areas of the genome. One of diabetic cases is caused by insulin resistance and changing the homeostasis of blood glucose control so glucose concentration stood beyond normal rate (hyperglycemia). High fat diet has been frequently studied and linked to triggering diabetes. However, most Asians consume rice (or food with high carbohydrate) and food with monosodium glutamate (MSG). This habit could lead to pathophysiology of type 2 diabetes mellitus (T2D). Previous studies showed that high-carbohydrate or high-MSG diet could change gene expression or modify protein activity in body metabolism. This imbalanced metabolism can lead to pleiotropic effects of diabetes mellitus. In this study, the authors have attempted to relate various changes in genes expression or protein activity to the high-carbohydrate and high-MSG-induced diabetes. The authors have also tried to relate several genes that contribute to pathophysiology of T2D and proposed several ideas of genes as markers and target for curing people with T2D. These are done by investigating altered activities of various genes that cause or are caused by diabetes. These genes are selected based on their roles in pathophysiology of T2D.

Aberrant PDK4 Promoter Methylation Preceding Hyperglycemia in a Mouse Model.

Diabetic prevalence is at speedy increase globally. Previous studies stated that other than genetics, factors such as environment, lifestyle, and paternal-maternal condition play critical roles in diabetes through DNA methylation in specific areas of the genome. The purpose of this study is to investigate the methylation pattern of the PDK4 promoter in streptozotocin-induced diabetic mice until the 12th week of the observation. The methylation pattern in the blood samples was analyzed periodically, while the pattern in the muscle sample was only analyzed at the end of the experiment using the blood of the sacrificed animals. Three methylated CpG site 1, CpG site 6, and CpG site 7 were analyzed and quantified based on the band density using bisulfite treatment and methylation-specific polymerase chain reaction (PCR). The hyperglycemia period was developed at the 9th week of experiment. However, there was a significant increase of methylation, specifically on CpG site 6 started from week 6 to week 12. This peculiar methylation on CpG site 6 of PDK4 promoter in the blood sample before the hyperglycemic period might serve as a potential biomarker for early detection of diabetes in the patients. No significant difference was found between the methylation level of streptozotocin (STZ)-treated mice and of the control group in the muscle sample.

Designing a less immunogenic nattokinase from Bacillus subtilis subsp. natto: a computational mutagenesis.

Nattokinase is an enzyme produced by Bacillus subtilis subsp. natto that contains strong fibrinolytic activity. It has potential to treat cardiovascular diseases. In silico analysis revealed that nattokinase is considered as an antigen, thus hindering its application for injectable therapeutic protein. Various web servers were used to predict B-cell epitopes of nattokinase both continuously and discontinuously to determine which amino acid residues had been responsible for the immunogenicity. With the exclusion of the predicted conserved amino acids, four amino acids such as S18, Q19, T242, and Q245 were allowed for mutation. Substitution mutation was done to lower the immunogenicity of native nattokinase. Through the stability of the mutated protein with the help of Gibbs free energy difference, the proposed mutein was S18D, Q19I, T242Y, and Q245W. The 3D model of the mutated nattokinase was modeled and validated with various tools. Physicochemical properties and stability analysis of the protein indicated that the mutation brought higher stability without causing any changes in the catalytic site of nattokinase. Molecular dynamics simulation implied that the mutation indicated similar stability, conformation, and behavior compared to the native nattokinase. These results are highly likely to contribute to the wet lab experiment to develop safer nattokinase.