Yu Chuan Huang

Detection of G-quadruplex DNA in mammalian cells

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

Immobilized DNA Switches as Electronic Sensors for Picomolar Detection of Plasma Proteins

The sensing principle of a new class of DNA conformational switches (deoxyribosensors) is based on the incorporation of an aptamer as the receptor, whose altered conformation upon analyte binding switches on the conductivity of an adjacent helical conduction path, leading to an increase in the measured electrical signal through the sensor. We report herein the rational design and biochemical testing of candidate deoxyribosensors for the detection and quantitation of a plasma protein, thrombin, followed by surface immobilization of the optimized sensor and its electrochemical testing in both a near-physiological buffer solution and in diluted blood serum. The very high detection sensitivity (in the picomolar range) and specificity, as well as the adaptability of deoxyribosensors for the detection of diverse molecular analytes both small and macromolecular, make this novel sensing methodology an extremely promising one. Such synthetic and robust DNA-based electronic sensors should find broad application in the rapid, miniaturized, and automated on-chip detection of many biomedically relevant substances (such as metabolites, toxins, and disease and tumor markers) as well as of environmental contaminants.

A Robust Electronic Switch Made of Immobilized Duplex/Quadruplex DNA†

In recent years, DNA and RNA have been used extensively as materials for the construction and self-assembly of a variety of nanoscale devices. DNA-based mechanical switches have been described recently. Extension–contraction transitions of DNA nanoconstructs (e.g., G-quadruplexes) upon ligand binding have also been reported. A G-quadruplex (G4DNA) is a quadruple-helical DNA secondary structure, in which guanines are paired by Hoogsteen hydrogen bonds to form guanine base quartets. G4-DNA is stabilized by the coordination of specific metal ions, such as K ions or Sr ions, which bind between successive G-quartets. G4-DNAbased nanoconstructs have been used as sensors to detect the blood protein thrombin, K ions, and target oligonucleotides by binding of these analytes to DNA aptamers (nucleic acid receptors that are themselves sometimes G4DNA). The ligand binding properties of G4-DNA, as well as its ability to conduct electrical charges, have been characterized extensively by using gel electrophoresis and spectroscopic methods. G4-DNAs typically form by the folding of G-rich singlestranded DNA (ssDNA), but such ssDNA to G4-DNA transitions are not easily reversed. In contrast, the duplex DNA (dsDNA) to G4-DNA transition can be kinetically more favored, brought about by the addition and removal of K ions (utilizing a chelator such as [18]crown-6). We recently reported a biochemical study of the charge-conducting properties of a contractile DNA nanoswitch, which is able to switch repeatedly between a structurally extended electronic “off” state (an extended duplex) and a contracted electronic “on” state (a G-quadruplex). The conductivity of the K-induced contracted duplex in solution was much higher than that of the extended conformational state. This biochemical study of conductivity, however, provided no direct indication of whether such a device could be adapted for practical use (i.e., whether electronic switching would still be observed if the device were to be localized on an electronic chip). Herein we report the results of our first chip-based strategy, which is able to directly and efficiently measure both the reversibility and the repeatability of the electronic switching behavior of the dsDNA/G4-DNA constructs. Specifically, we present an electrochemical (square-wave voltammetry) investigation of the DNA nanoswitch immobilized on a gold electrode. As shown in Figure 1, the contractile DNA duplex incorporates two short, separated motifs of G/G mismatches that are flanked by Watson–Crick base-paired DNA. The

Charge Conduction Properties of a Parallel-Stranded DNA G-Quadruplex: Implications for Chromosomal Oxidative Damage

The charge-flow properties and concomitant guanine damage patterns of a number of intermolecular and wholly parallel-stranded DNA G-quadruplexes were investigated. The DNA constructs were structurally well-defined and consisted of the G-quadruplex sandwiched and stacked between two Watson-Crick base-paired duplexes. Such duplex-quadruplex-duplex constructs were designed to minimize torsional stress as well as steric crowding at the duplex-quadruplex junctions. When anthraquinone was used to induce charge flow within the constructs, it was found that the quadruplex served both as a sink and as a moderately good conductor of electron holes, relative to DNA duplexes. Most strikingly, the quadruplex suffered very little charge-flow generated oxidative damage relative to guanines in the duplex regions and, indeed, to guanines in antiparallel quadruplexes reported in prior studies. It is likely that these differences result from a combination of steric and electronic factors. A biological conclusion that may be drawn from these data is that if, as anticipated, G-quadruplex structures form in vivo at the telomeres and other loci in eukaryotic chromosomes, their ability to serve as protective sinks against chromosomal oxidative damage may depend on their specific character and topology. From a separate perspective, our results on the conduction properties of duplex-quadruplex-duplex DNA composites suggest the utility of G-quadruplexes as junction modules in the construction of DNA-based biosensors and nanocircuitry.

Heme activation by DNA: isoguanine pentaplexes, but not quadruplexes, bind heme and enhance its oxidative activity

Guanine-rich, single-stranded, DNAs and RNAs are able to fold to form G-quadruplexes that are held together by guanine base quartets. G-quadruplexes are known to bind ferric heme [Fe(III)-protoporphyrin IX] and to strongly activate such bound hemes toward peroxidase (1-electron oxidation) as well as oxygenase/peroxygenase (2-electron oxidation) activities. However, much remains unknown about how such activation is effected. Herein, we investigated whether G-quadruplexes were strictly required for heme activation or whether related multi-stranded DNA/RNA structures such as isoguanine (iG) quadruplexes and pentaplexes could also bind and activate heme. We found that iG-pentaplexes did indeed bind and activate heme comparably to G-quadruplexes; however, iG-quadruplexes did neither. Earlier structural and computational studies had suggested that while the geometry of backbone-unconstrained iG-quintets templated by cations such as Na+ or NH4+ was planar, that of iG-quartets deviated from planarity. We hypothesize that the binding as well as activation of heme by DNA or RNA is strongly supported by the planarity of the nucleobase quartet or quintet that interacts directly with the heme.

A Twisting Electronic Nanoswitch Made of DNA

Single-stranded DNAs and RNAs that are rich in the nucleobase guanine form four-stranded G-quadruplexes, which are held together by hydrogen-bonded guanine quartets. In aqueous solution, both DNA duplexes and G-quadruplexes are modest conductors of electrical charge. A tight, topologically constrained DNA construct called twDNA is now reported, in which a core of four guanine-rich single strands structurally and electronically links together four DNA double helices. The addition and removal of K(+) or Sr(2+) cations promote alternative conformers of twDNA, which have strikingly distinct electronic properties. Unlike DNA mechano-electronic switches that require large conformational changes, twDNA requires only modest twisting/untwisting structural attenuations to achieve electronic switching.

Mechatronic DNA devices driven by a G-quadruplex-binding platinum ligand

Contractile duplexes are DNA double helices that incorporate two strategically placed patches of guanine-guanine (G·G) base mismatches. Such duplexes are cation-driven mechatronic devices, able to toggle between states with distinct mechanical and charge conduction properties. In aqueous lithium chloride solution contractile duplexes have an extended (E) and poorly conductive conformation; however, potassium ions drive them to a relatively conductive and structurally contracted (C) conformation, via intramolecular G-quadruplex formation. Here, we report that even in the absence of K(+) ions, a known G-quadruplex binding ligand, Pt-PIP [phenylphenanthroimidazole ethylenediamine platinum(II)] efficiently promotes the E→C transition, while a poor binder, Pt-bpy [bipyridine ethylenediamine platinum(II)], does not promote this transition. An examination of E→C transitions within two different designs for DNA contractile helices found an unexpected complexity: the formation of distinct C states, both electrically conductive, but possessing dissimilar DNA topologies. Ligand-driven DNA mechatronic devices such as these may constitute prototypes for electronic biosensors that identify G-quadruplex binding ligands.

Erratum: Expression of concern: Detection of G-quadruplex DNA in mammalian cells

1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada, 2Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, NIH Biomedical Research Center, Baltimore, MD 21224, USA, 3Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada, 4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104-6100, USA, 5Division of Hematology, Department of Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada and 6European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands

DNA mechatronic devices switched by K+ and by Sr2+ are structurally, topologically, and electronically distinct

DNAs and RNAs that fold via the formation of guanine quartets form G‐quadruplexes that are often highly diverse in terms of architecture and topology. G‐quadruplexes are specifically stabilized by metal cations such as K+ and Sr2+, but not Li+. DNA duplexes that incorporate two separated clusters of G•G mismatches (“P‐duplexes”) can function as electronic switches, capable of toggling reversibly from a poorly conductive conformer (E) with only Li+ in the solution to a G‐quadruplex incorporating conformer of higher conductivity (C) in the presence of K+. Herein, we report results from fluorescence energy transfer, circular dichroism, charge conduction, and chemical footprinting experiments, which cumulatively demonstrate that P‐duplex E↔C transitions are genuinely mechatronic, with causally coupled mechanical and electronic states. We show, further, that the K+‐ and the Sr2+‐fuelled E↔C switching of a given P‐duplex are structurally, topologically, and electronically distinct from each other. A single DNA P‐duplex can thus exist in at least three distinguishable mechatronic states in aqueous solution.