Methods for investigating G-quadruplex DNA/ligand interactions

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.     

Ligand Design to Acquire Specificity to Intended G-Quadruplex Structures

A G-quadruplex is a nucleic acid secondary structure that is adopted by guanine-rich sequences, and is considered to be relevant in various pharmacological and biological contexts. G-Quadruplexes have also attracted great attention in the field of DNA nanotechnology because of their extremely high thermal stability and the availability of many defined structures. To date, a large repertory of DNA/RNA G-quadruplex-interactive ligands has been developed by numerous laboratories. Several relevant reviews have also been published that have helped researchers to grasp the full scope of G-quadruplex research from its outset to the present. This review focuses on the G-quadruplex ligands that allow targeting of specific G-quadruplexes. Moreover, unique ligands, successful methodologies, and future perspectives in relation to specific G-quadruplex recognition are also addressed.     

特异性识别DNA的吡咯-咪唑多聚酰胺的研究进展

吡咯-咪唑多聚酰胺为一类人工合成的主要由五元杂环化合物N-甲基吡咯(Py)、N-甲基咪唑(Im)和N-甲基3-羟基吡咯(Hp)芳香氨基酸组成的,经酰胺键连接的人工小分子配体.它们具有与天然DNA结合蛋白相媲美的DNA特异性识别和结合能力.近20年来,对此类化合物的研究取得了重要进展,确定了简单的氨基酸对识别碱基对的规则,研究了多种方式连接的吡咯-咪唑多聚酰胺与DNA小沟结合模式,合成了多种双功能吡咯-咪唑多聚酰胺,且吡咯-咪唑多聚酰胺能穿过细胞膜,具有在体内外调节基因表达的作用。     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

Metallo-intercalators and metallo-insertors

Since the elucidation of the structure of double helical DNA, the construction of small molecules that recognize and react at specific DNA sites has been an area of considerable interest. In particular, the study of transition metal complexes that bind DNA with specificity has been a burgeoning field. This growth has been due in large part to the useful properties of metal complexes, which possess a wide array of photophysical attributes and allow for the modular assembly of an ensemble of recognition elements. Here we review recent experiments in our laboratory aimed at the design and study of octahedral metal complexes that bind DNA non-covalently and target reactions to specific sites. Emphasis is placed both on the variety of methods employed to confer site-specificity and upon the many applications for these complexes. Particular attention is given to the family of complexes recently designed that target single base mismatches in duplex DNA through metallo-insertion.     

Synthetic peptides in the diagnosis of systemic autoimmune diseases

Proteins recognized by antibodies from patients with autoimmune diseases have been intensively studied over the two past decades since cDNAs encoding autoantigens have become available. Identity of many of them has been defined, and specific structural motifs or post-translational modifications, which may be important to explain the generation of such antibodies during the autoimmune process, have been pointed out. Immunological analysis of sera from autoimmune patients with recombinant fragments and with short peptides has revealed the presence of dominant epitopes along proteins; some of them are targeted by antibodies from patients with specific diseases or disease subsets. Innovative technologies such as peptide arrays and biosensors as well as the exploitation of large peptides libraries have recently open up new perspectives. Peptides bearing natural modifications, peptide analogues, as well as mimotopes of protein or non-protein antigens (DNA, RNA, sugar) have been developed and might advantageously replace native antigens in routine immunoassays. Although numerous conformational epitopes have not yet been identified, and cannot be identified by the approaches classically used in epitope mapping studies, such peptides and peptide analogues may represent efficient probes to detect the presence of circulating autoantibodies in the serum of autoimmune patients and help for establishing specific and sensitive early diagnostic tests. They may also lead to the design of high-affinity ligands for purifying autoantibodies. These different aspects are discussed and epitope mapping studies of a number of autoantigens (e.g. histones, sn and hnRNP proteins and Ro proteins) are summarized.     

Nucleic Acid Nanostructures and Topology

Knots, polyhedra, and Borromean rings with specific structural and topological features can be made from DNA. Biotechnologists have been exploiting the programmability of DNA intermolecular associations for a quarter of a century. These operations have now been applied successfully to branched DNA species to produce complex target structures (for example, the cube shown in the picture) and a nanomechanical device. The assembly of two-dimensional crystals with programmed topographic characteristics demonstrates the simplicity of translating design into surface structures.     

Natural products and their derivatives as G-quadruplex binding ligands

G-quadruplexes comprise a class of secondary structures that are formed in guanine-rich sequences in eukaryotic genomes and play a crucial role in the regulation of many biological events. G-quadruplexes have become targets for anticancer drugs with high selectivity vs. duplex DNA and low cytotoxicity against normal cells. Natural products and their derivatives display polymorphism, structural complexity, and potent activity. It is, therefore, reasonable to seek ligands targeting G-quadruplexes from natural products. Recently, many successful examples have been reported, showing ligands with excellent anticancer activities. In this review, we summarized the development of research on natural products and derivatives that target G-quadruplex structures in an effort to guide future studies.     

Aptamer–nanoparticle complexes as powerful diagnostic and therapeutic tools

Correct diagnosis and successful therapy are extremely important to enjoy a healthy life when suffering from a disease. To achieve these aims, various cutting-edge technologies have been designed and fabricated to diagnose and treat specific diseases. Among these technologies, aptamer–nanomaterial hybrids have received considerable attention from scientists and doctors because they have numerous advantages over other methods, such as good biocompatibility, low immunogenicity and controllable selectivity. In particular, aptamers, oligonucleic acids or peptides that bind to a specific target molecule, are regarded as outstanding biomolecules. In this review, several screening techniques for aptamers, also called systematic evolution of ligands by exponential enrichment (SELEX) methods, are introduced, and diagnostic and therapeutic aptamer applications are also presented. Furthermore, we describe diverse aptamer–nanomaterial conjugate designs and their applications for diagnosis and therapy.     

NDI-induced Topological Conversion of Human Telomeric G-Quadruplexes from Hybrid-2 to Parallel Form

G-Quadruplexes(GQs), which are formed by G-rich DNA sequences in human telomeres, have become an attractive target for cancer treatment. The ligands to stabilize the conformation of human telomeric GQs in vivo are particularly important for structure-based ligand design and drug development targeting the noncanonical DNA structure. Here we report the conformational conversion of Tel26 induced by a naphthalene diimide(NDI) ligand in K⁺ buffer, even at cellular physiological temperature(37℃) and under mimetic cellular crowding conditions created by Ficoll 70. We provide an insight into the dynamic conversion from initial hybrid-2 GQ topology to final parallel GQ topology. These results are helpful for the design of ligands with GQ conformation regulation.     

Self-quenching smart probes as a platform for the detection of sequence-specific UV-induced DNA photodamage

Molecular beacons (MBs) are sensitive probes for many DNA sequence-specific applications, such as DNA damage detection, but suffer from technical and cost limitations. We have designed smart probes with self-quenching properties as an alternative to molecular beacons to monitor sequence-specific UV-induced photodamage of oligonucleotides. These probes have similar stem-loop structural characteristics as molecular beacons, but quenching is achieved instead via photoinduced intramolecular electron transfer by neighboring guanosine residues. Our results indicate that the probes are sensitive enough to detect nanomolar target concentrations and are specific enough to discriminate single-base damage. When the probes were used to monitor UV-induced photodamage in oligonucleotide sequences that differ by a single-base mismatch, the photodamage time constant was higher for the perfectly complementary target sequences than for the mismatch sequences, indicating that these probes are specific for each target sequence. In addition, time constants obtained for oligonucleotide target sequences with both stem and loop base mismatches are lower than those with only loop mismatches, suggesting that these sequences are also specifically distinguished by the smart probes. These probes thus constitute robust, sensitive, specific, and cheaper alternatives to MBs for sequence-specific DNA damage detection.     

Recent advances in fluorescent probes for the detection of reactive oxygen species

Reactive oxygen species (ROS) have captured the interest of many researchers in the chemical, biological, and medical fields since they are thought to be associated with various pathological conditions. Fluorescent probes for the detection of ROS are promising tools with which to enhance our understanding of the physiological roles of ROS, because they provide spatial and temporal information about target biomolecules in in vivo cellular systems. ROS probes, designed to detect specific ROS with a high selectivity, would be desirable, since it is now becoming clear that each ROS has its own unique physiological activity. However, dihydro-compounds such as 2′,7′-dichlorodihydrofluorescein (DCFH), which have traditionally been used for detecting ROS, tend to react with a wide variety of ROS and are not completely photostable. Some attractive fluorescent probes that exhibit a high degree of selectivity toward specific ROS have recently been reported, and these selective probes are expected to have great potential for elucidating unknown physiological mechanisms associated with their target ROS. This review focuses on the design, detection mechanism, and performance of fluorescent probes for the detection of singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radicals (^.OH), or superoxide anion (O₂ ^−.), a field in which remarkable progress has been achieved in the last few years.     

An ontology for pharmaceutical ligands and its application for in silico screening and library design

Annotation efforts in biosciences have focused in past years mainly on the annotation of genomic sequences. Only very limited effort has been put into annotation schemes for pharmaceutical ligands. Here we propose annotation schemes for the ligands of four major target classes, enzymes, G protein-coupled receptors (GPCRs), nuclear receptors (NRs), and ligand-gated ion channels (LGICs), and outline their usage for in silico screening and combinatorial library design. The proposed schemes cover ligand functionality and hierarchical levels of target classification. The classification schemes are based on those established by the EC, GPCRDB, NuclearDB, and LGICDB. The ligands of the MDL Drug Data Report (MDDR) database serve as a reference data set of known pharmacologically active compounds. All ligands were annotated according to the schemes when attribution was possible based on the activity classification provided by the reference database. The purpose of the ligand-target classification schemes is to allow annotation-based searching of the ligand database. In addition, the biological sequence information of the target is directly linkable to the ligand, hereby allowing sequence similarity-based identification of ligands of next homologous receptors. Ligands of specified levels can easily be retrieved to serve as comprehensive reference sets for cheminformatics-based similarity searches and for design of target class focused compound libraries. Retrospective in silico screening experiments within the MDDR01.1 database, searching for structures binding to dopamine D2, all dopamine receptors and all amine-binding class A GPCRs using known dopamine D2 binding compounds as a reference set, have shown that such reference sets are in particular useful for the identification of ligands binding to receptors closely related to the reference system. The potential for ligand identification drops with increasing phylogenetic distance. The analysis of the focus of a tertiary amine based combinatorial library compared to known amine binding class A GPCRs, peptide binding class A GPCRs, and LGIC ligands constitutes a second application scenario which illustrates how the focus of a combinatorial library can be treated quantitatively. The provided annotation schemes, which bridge chem- and bioinformatics by linking ligands to sequences, are expected to be of key utility for further systematic chemogenomics exploration of previously well explored target families.     

Fluorescence light-up recognition of DNA nucleotide based on selective abasic site binding of an excited-state intramolecular proton transfer probe

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation-related diseases. Various fluorescence methods for SNP detection have been proposed and many are already in use. However, fluorescence enhancement for signal-on SNP identification without label modification still remains a challenge. Here, we find that the abasic site (AP site) in a DNA duplex can be developed as a binding pocket favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of a 3-hydroxyflavone, fisetin, which is used as a proof of concept for effective SNP identification. Fisetin binding at the AP site is highly selective for target thymine or cytosine facing the AP site by observation of a drastic increase in the ESIPT emission band. In addition, the target recognition selectivity based on this ESIPT process is not affected by flanking bases of the AP site. The binding selectivity of fisetin at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, emission lifetime and DNA melting. The fluorescent signal-on sensing for SNP based on this fluorophore is substantially advantageous over the previously used fluorophores such as the AP site-specific signal-off organic ligands with a similar fluorescing mechanism before and after binding to DNA with hydrogen bonding interaction. We expect that this approach will be employed to develop a practical SNP detection method by locating an AP site toward a target and employing an ESIPT probe as readout.     

Covalent strategy for immobilization of DNA-microspots suitable for microarrays with label-free and time-resolved optical detection of hybridization

Sequence-specific detection and quantification of nucleic acids are central steps in many molecular biology procedures which have also been transferred to chip-based procedures. Hybridization-based assays can be used to quantify and discriminate between DNA target sequences down to the level of single base mismatches. Arrays of DNA probes immobilized on a support enable simultaneous testing of multiple sequences of a single sample. DNA arrays can be produced either by in-situ synthesis of oligonucleotides or by immobilization of pre-assembled DNA probes. Covalent and directed immobilization improves the reproducibility and stability of DNA arrays. This is especially interesting with repeated use of transducers or chips. Procedures are described for effective covalent immobilization of pre-assembled amino-linked oligonucleotides, by use of ink-jet techniques, on a modified and heated glass surface, with addressable surface areas ranging from 0.01 mm2 to a few mm2. Almost immediate evaporation of the spotted droplets on the heated surfaces leads to very high surface hybridization capacities. The surfaces are suitable for use with a label-free detection method - reflectometric interference spectroscopy (RIfS). It is shown that hybridization capacity and non-specific interaction at these DNA-surfaces can be characterized by use of RIfS. With a consumption of less than 80 ng mm(-2) oligonucleotide and a specific hybridization capacity of more than 300 fmol mm(-2), the activated aminodextran procedure was usually suitable for setting up a DNA array with label-free detection. Non-specific interactions with random oligomers or protein (ovalbumin) were low. Up to 150 repeated regenerations (stripping) of the surfaces by acid treatment and denaturing agents, and 50 days of storage, have been possible without significant loss of hybridization capacity.     

A practical approach to screen for authorised and unauthorised genetically modified plants

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 × 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.