Structural varieties of selectively mixed G‐ and C‐rich short DNA sequences studied with electrospray ionization mass spectrometry

Short guanine(G)‐repeat and cytosine(C)‐repeat DNA strands can self‐assemble to form four‐stranded G‐quadruplexes and i‐motifs, respectively. Herein, G‐rich and C‐rich strands with non‐G or non‐C terminal bases and different lengths of G‐ or C‐repeats are mixed selectively in pH 4.5 and 6.7 ammonium acetate buffer solutions and studied by electrospray ionization mass spectrometry (ESI‐MS). Various strand associations corresponding to bi‐, tri‐ and tetramolecular ions are observed in mass spectra, indicating that the formation of quadruplex structures is a random strand by strand association process. However, with increasing incubation time for the mixtures, initially associated hybrid tetramers will transform into self‐assembled conformations, which is mainly driven by the structural stability. The melting temperature values of self‐assembled quadruplexes suggest that the length of G‐repeats or C‐repeats shows more significant effect on the stability of quadruplex structures than that of terminal residues. Accordingly, we can obtain the self‐associated tetrameric species generated from the mixtures of various homologous G‐ or C‐strands efficiently by altering the length of G‐ or C‐repeats. Our studies demonstrate that ESI‐MS is a very direct, fast and sensitive tool to provide significant information on DNA strand associations and stoichiometric transitions, particularly for complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.     

Targeting Non‐B‐Form DNA in Living Cells

Under certain conditions, repetitive DNA motifs have the potential to adopt non‐B‐form DNA structures, such as hairpins, triplexes, Z‐DNA, quadruplexes, and i‐motifs. Some non‐B‐form DNAs have been proposed to cause mutations and, consequently, participate in several biologically important processes, including regulation, evolution, and human disease. Advancement in the knowledge of specific interactions between molecules and non‐B‐form DNAs at the molecular level in living cells is important for understanding their biological functions. In this review, we describe the latest studies on molecules that target non‐B‐form DNAs in vivo, with a focus on Z‐DNA, G‐quadruplexes, triplexes, i‐motifs, and hairpins.     

Monomer–Dimer Equilibrium for the 5′–5′ Stacking of Propeller‐Type Parallel‐Stranded G‐Quadruplexes: NMR Structural Study

Guanine‐rich sequence motifs, which contain tracts of three consecutive guanines connected by single non‐guanine nucleotides, are abundant in the human genome and can form a robust G‐quadruplex structure with high stability. Herein, by using NMR spectroscopy, we investigate the equilibrium between monomeric and 5′–5′ stacked dimeric propeller‐type G‐quadruplexes that are formed by DNA sequences containing GGGT motifs. We show that the monomer–dimer equilibrium depends on a number of parameters, including the DNA concentration, DNA flanking sequences, the concentration and type of cations, and the temperature. We report on the high‐definition structure of a simple monomeric G‐quadruplex containing three single‐residue loops, which could serve as a reference for propeller‐type G‐quadruplex structures in solution.     

Highly Reactive and Tracelessly Cleavable Cysteine‐Specific Modification of Proteins via 4‐Substituted Cyclopentenone

A rapid and cysteine‐specific modification of proteins using 4‐substituted cyclopentenone via a Michael addition tandem elimination reaction was developed. Compared to the classical method, this reaction featured fast kinetics with a stable product. More importantly, this conjugation could be tracelessly removed by exchange with a Michael addition donor. The conjugation and regeneration process not only exhibited little change to the structures or conformations of the proteins but also exhibited little disturbance to their biological functions, such as their enzymatic activities.     

AIEgens/Nucleic Acid Nanostructures for Bioanalytical Applications

DNA occupies significant roles in life processes, which include encoding the sequences of proteins and accurately transferring genetic information from generation to generation. Recent discoveries have demonstrated that a variety of biological functions are correlated with DNA′s conformational transitions. The non‐B form has attained great attention among the diverse forms of DNA over the past several years. The main reason for this is that a large number of studies have shown that the non‐B form of DNA is associated with gross deletions, inversions, duplications, translocations as well as simple repeating sequences, which therefore causes human diseases. Consequently, the conformational transition of DNA between the B‐form and the non‐B form is important for biology. Conventional fluorescence probes based on the conformational transitions of DNA usually need a fluorophore and a quencher group, which suffers from the complex design of the structure and tedious synthetic procedures. Moreover, conventional fluorescence probes are subject to the aggregation‐caused quenching (ACQ) effect, which limits their application toward imaging and analyte detection. Fluorogens exhibiting aggregation‐induced emission (AIE) have attracted tremendous attention over the past decade. By taking advantage of this unique behavior, plenty of fluorescent switch‐on probes without the incorporation of fluorescent quenchers/fluorophore pairs have been widely developed as biosensors for imaging a variety of analytes. Herein, the recent progress in bioanalytical applications on the basis of aggregation‐induced emission luminogens (AIEgens)/nucleic acid nanostructures are presented and discussed.     

Reversibility of the low‐temperature transitions of polytetrafluoroethylene as revealed by temperature‐modulated differential scanning calorimetry

Temperature‐modulated differential scanning calorimetry reveals distinct differences in the kinetics of the low‐temperature phase transitions of polytetrafluoroethylene. The triclinic to trigonal transition at 292 K is partially reversible as long it is not complete. As soon as the total sample is converted, supercooling is required to nucleate the reversal of the helical untwisting involved in the transition. The trigonal phase can be annealed in the early stages after transformation with a relaxtion time of about 5 minutes. The dependence of the reversing heat capacity on the modulation amplitude, after a metastable equilibrium has been reached, is explained by a non‐linear, time‐independent increase of the heat‐flow rate, perhaps caused by an increased true heat capacity. The order‐disorder‐transition at 303 K from the trigonal to a hexagonal condis phase is completely reversible and time‐independent. It extends to temperatures as low as the transition at 292 K or even lower. Qualitatively, the thermal history and crystallization conditions of polytetrafluoroethylene do not affect the transition kinetics, that is, melt‐crystallized film and as‐polymerized powders show similar transition behaviors, despite largely different crystallinities. © 2001 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 750–756, 2001     

Multistep conformational interconversion mechanism of cyclododecane. A simple and fast analysis using potential energy curves

An ab initio and Density Functional Theory (DFT) study of the conformational properties of cyclododecane was carried out. The energetically preferred equilibrium structures, their relative stability, and some of the transition state (TS) structures involved in the conformational interconversion pathways were analyzed from RHF/6‐31G(d), B3LYP/6‐31G(d,p) and B3LYP/6311++G(d,p) calculations. Aug‐cc‐pVDZ//B3LYP/6311++G(d,p) single point calculations predict that the multistep conformational interconversion mechanism requires 11.07 kcal/mol, which is in agreement with the available experimental data. These results allow us to form a concise idea about the internal intricacies of the preferred forms of cyclododecane, describing the conformations as well as the conformational interconversion processes in the conformational potential energy hypersurface. Our results indicated that performing an exhaustive analysis of the potential energy curves connecting the most representative conformations is a valid alternate tool to determine the principal conformational interconversion paths for cyclododecane. This methodology represents a satisfactory first approximation for the conformational analysis of medium‐ and large‐size flexible cyclic compounds. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2012     

A Usual G‐Protein‐Coupled Receptor in Unusual Membranes

G‐protein‐coupled receptors (GPCRs) are the largest family of membrane‐bound receptors and constitute about 50 % of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge‐interaction‐directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid‐ and polymer‐based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non‐biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H⁺] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin‐conserved ERY sequence motif that helps break the cytoplasmic “ionic lock”. This study unveils a novel design concept of non‐biological membranes to reconstitute and harness GPCR functions in synthetic systems.     

Pyridyl‐Substituted Corrole Isomers: Synthesis and their Regulation to G‐quadruplex Structures

G‐quadruplex DNA plays an important role in the potential therapeutic target for the design and development of anticancer drugs. As various G‐quadruplex sequences in the promoter regions or telomeres can form different secondary structural modes and display a diversity of biology functions, variant G‐quadruplex interactive agents may be necessary to cure different disease by differentiating variant types of G‐quadruplexes. We synthesize five cationic methylpyridylium corroles and compare the interactions of corroles with different types of G‐quadruplexes such as cmyc, htelo, and bcl2 by using surface plasmon resonance. Because of the importance of human telomere G‐quadruplex DNA, we focus on the biological properties of the interactions between human telomere G‐quadruplex DNA and corrole isomers using CD, T_m, PCR‐stop (PCR= polymerase chain reaction), and polymerase‐stop assay, which demonstrate the excellent ability of the corrole to induce and stabilize the G‐quadruplex. This study provides the first experimental insight into how selectivity might be achieved for different G‐quadruplexes by a single group of methylpyridylium corrole isomers that may be optimized for potential selective cancer therapy.     

Collisional and Coulombic Unfolding of Gas‐Phase Proteins: High Correlation to Their Domain Structures in Solution

The three‐dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher‐order assemblies. While important coursed‐grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas‐phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility‐mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. This correlation and its potential uses for structural biology is discussed.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.     

An Aggregating Amphiphilic Squaraine: A Light‐up Probe That Discriminates Parallel G‐Quadruplexes

G‐quadruplexes (G4s) are peculiar DNA or RNA tertiary structures that are involved in the regulation of many biological events within mammalian cells, bacteria, and viruses. Although their role as versatile therapeutic targets has been emphasized for 35 years, G4 selectivity over ubiquitous double‐stranded DNA/RNA, as well as G4 differentiation by small molecules, still remains challenging. Here, a new amphiphilic dicyanovinyl‐substituted squaraine, SQgl , is reported to act as an NIR fluorescent light‐up probe discriminating an extensive panel of parallel G4s while it is non‐fluorescent in the aggregated state. The squaraine can form an unconventional sandwich π‐complex binding two quadruplexes, which leads to a strongly fluorescent (Φ _F=0.61) supramolecular architecture. SQgl is highly selective against non‐quadruplex and non‐parallel G4 sequences without altering their topology, as desired for applications in selective in vivo high‐resolution imaging and theranostics.     

Quantum mechanical studies on model α‐pleated sheets

Pauling and Corey proposed a pleated‐sheet configuration, now called α‐sheet, as one of the protein secondary structures in addition to α‐helix and β‐sheet. Recently, it has been suggested that α‐sheet is a common feature of amyloidogenic intermediates. We have investigated the stability of antiparallel β‐sheet and two conformations of α‐sheet in solution phase using the density functional theoretical method. The peptides are modeled as two‐strand acetyl‐(Ala)₂‐N‐methylamine. Using stages of geometry optimization and single point energy calculation at B3LYP/cc‐pVTZ//B3LYP/6‐31G* level and including zero‐point energies, thermal, and entropic contribution, we have found that β‐sheet is the most stable conformation, while the α‐sheet proposed by Pauling and Corey has 13.6 kcal/mol higher free energy than the β‐sheet. The α‐sheet that resembles the structure observed in molecular dynamics simulations of amyloidogenic proteins at low pH becomes distorted after stages of geometry optimization in solution. Whether the α‐sheets with longer chains would be increasingly favorable in water relative to the increase in internal energy of the chain needs further investigation. Different from the quantum mechanics results, AMBER parm94 force field gives small difference in solution phase energy between α‐sheet and β‐sheet. The predicted amide I IR spectra of α‐sheet shows the main band at higher frequency than β‐sheet. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Heat‐Flow‐Driven Oligonucleotide Gelation Separates Single‐Base Differences

DNA phase transitions are often induced by the addition of condensation agents or by dry concentration. Herein, we show that the non‐equilibrium setting of a moderate heat flow across a water‐filled chamber separates and gelates DNA strands with single‐base resolution. A dilute mix of DNA with two slightly different gel‐forming sequences separates into sequence‐pure hydrogels under constant physiological solvent conditions. A single base change in a 36 mer DNA inhibits gelation. Only sequences with the ability to form longer strands are concentrated, further elongated, and finally gelated by length‐dependent thermal trapping. No condensation agents, such as multivalent ions, were added. Equilibrium aggregates from dry concentration did not show any sequence separation. RNA is expected to behave identically owing to its equal thermophoretic properties. The highly sequence‐specific phase transition points towards new possibilities for non‐equilibrium origins of life.     

Surface‐Assisted Large‐Scale Ordering of DNA Origami Tiles

The arrangement of DNA‐based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close‐packing of symmetric, non‐interacting DNA origami structures, or by utilizing blunt‐end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.     

Peptide models XXXI. Conformational properties of hydrophobic residues shaping the core of proteins. An ab initio study of N‐formyl‐L‐valinamide and N‐formyl‐L‐phenylalaninamide

Employing introductory (3‐21G RHF) and medium‐size (6‐311++G** B3LYP) ab initio calculations, complete conformational libraries, containing as many as 27 conformers, have been determined for diamide model systems incorporating the amino acids valine (Val) and phenylalanine (Phe). Conformational and energetic properties of these libraries were analyzed. For example, significant correlation was found between relative energies from 6‐311++G** B3LYP and single‐point B3LYP/6‐311++G**//RHF/3‐21G calculations. Comparison of populations of molecular conformations of hydrophobic aromatic and nonaromatic residues, based on their ab initiorelative energies, with their natural abundance indicates that, at least for the hydrophobic core of proteins, the conformations of Val (Ile, Leu) and Phe (Tyr, Trp) are controlled by the local energetic preferences of the respective amino acids. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 732–751, 2001     

Orienting Tetramolecular G‐Quadruplex Formation: The Quest for the Elusive RNA Antiparallel Quadruplex

DNA and RNA G‐quadruplexes (G4) are unusual nucleic acid structures involved in a number of key biological processes. RNA G‐quadruplexes are less studied although recent evidence demonstrates that they are biologically relevant. Compared to DNA quadruplexes, RNA G4 are generally more stable and less polymorphic. Duplexes and quadruplexes may be combined to obtain pure tetrameric species. Here, we investigated whether classical antiparallel duplexes can drive the formation of antiparallel tetramolecular quadruplexes. This concept was first successfully applied to DNA G4. In contrast, RNA G4 were found to be much more unwilling to adopt the forced antiparallel orientation, highlighting that the reason RNA adopts a different structure must not be sought in the loops but in the G‐stem structure itself. RNA antiparallel G4 formation is likely to be restricted to a very small set of peculiar sequences, in which other structural features overcome the formidable intrinsic barrier preventing its formation.     

A density functional theory study on diazotization and nitration of 3,5‐diamino‐1,2,4‐triazole

The reaction mechanism, thermodynamic and kinetic properties for diazotization and nitration of 3,5‐diamino‐1,2,4‐triazole were studied by a density functional theory. The geometries of the reactants, transition states, and intermediates were optimized at the B3LYP/6‐31G (d, p) level. Vibrational analysis was carried out to confirm the transition state structures, and the intrinsic reaction coordinate (IRC) method was used to explore the minimum energy path. The single‐point energies of all stagnation points were further calculated at the B3LYP (MP2)/6‐311+G (2d, p) level. The statistical thermodynamic method and Eyring transition state theory with Wigner correction were used to study the thermodynamic and kinetic characters of all reactions within 0–25°C. Two reaction channels are computed, including the diazotization and nitration of 3‐NH₂ or 5‐NH₂, and there are six steps in each channel. The reaction rate in each step is increased with temperature. The last step in each channel is the slowest step. The first, second, and fifth steps are exothermic reactions, and are favored at lower temperature in the thermodynamics. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011