Short DNAzymes for Efficient Catalysis of "Click" Reactions in Solution and on Surface.

We have systematically investigated and found surprising superior catalytic activities of very short DNAzymes for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), both in solution and on surface. As a key reaction of the "click chemistry" class, CuAAC is a highly efficient and specific covalent conjugation tool with demonstrated applications in organic synthesis, bioconjugation, and surface functionalization; however, it requires the presence of the Cu(I) catalyst, which is an unstable species in aqueous solutions. We show here that one ultrashort, 14-nucleotide-truncated fragment of an earlier in vitro selected DNAzyme (CLICK-17) shows a striking and superior catalytic activity toward the in trans CuAAC reaction in solution and on surface in the presence of either Cu(I) or Cu(II), at significantly lowered concentrations. These results obviate the need for long-sequence DNAzymes, selected out of the homogeneous solution phase, for application in complex surface environments.

Unusual Paradigm for DNA-DNA Recognition and Binding: "Socket-Plug" Complementarity.

DNA is the key informational polymer in biology by virtue of its precisely defined self-assembling properties. Watson-Crick complementarity, which underlies DNA's self-assembly, is required not only in biology but has also proved powerful in the field of nanoscience, where it has been utilized to assemble complex 2D and 3D architectures and nanodevices built from the DNA double-helix. Aside from Watson-Crick base-pairing, however, DNA also participates in alternative base pairing schemes, giving rise to DNA triplexes and G-quadruplexes. Herein, we describe "sticky-ended" DNA triplex-quadruplex composites that specifically recognize and bind to each other using a wholly different logic, "socket-plug" complementarity, a shape-sensing fitting of guanine "prongs" into guanine-lacking "cavities." A remarkable property of this kind of complementarity is the key role played in it by specific counter-cations: thus, exclusive "self" socket-plug recognition occurs over "other" in sodium salt solutions while precisely the reverse occurs in potassium salt solutions. We have used gel electrophoresis, Förster resonance energy transfer, alkylation protection, and structural modeling to study this remarkable fundamental property of DNA, that we anticipate will find wide practical application.

A heme•DNAzyme activated by hydrogen peroxide catalytically oxidizes thioethers by direct oxygen atom transfer rather than by a Compound I-like intermediate.

Hemin [Fe(III)-protoporphyrin IX] is known to bind tightly to single-stranded DNA and RNA molecules that fold into G-quadruplexes (GQ). Such complexes are strongly activated for oxidative catalysis. These heme•DNAzymes and ribozymes have found broad utility in bioanalytical and medicinal chemistry and have also been shown to occur within living cells. However, how a GQ is able to activate hemin is poorly understood. Herein, we report fast kinetic measurements (using stopped-flow UV-vis spectrophotometry) to identify the H2O2-generated activated heme species within a heme•DNAzyme that is active for the oxidation of a thioether substrate, dibenzothiophene (DBT). Singular value decomposition and global fitting analysis was used to analyze the kinetic data, with the results being consistent with the heme•DNAzyme's DBT oxidation being catalyzed by the initial Fe(III)heme-H2O2 complex. Such a complex has been predicted computationally to be a powerful oxidant for thioether substrates. In the heme•DNAzyme, the DNA GQ enhances both the kinetics of formation of the active intermediate as well as the oxidation step of DBT by the active intermediate. We show, using both stopped flow spectrophotometry and EPR measurements, that a classic Compound I is not observable during the catalytic cycle for thioether sulfoxidation.

Non-Destructive Direct Pericarp Thickness Measurement of Sorghum Kernels Using Extended-Focus Optical Coherence Microscopy.

Non-destructive measurements of internal morphological structures in plant materials such as seeds are of high interest in agricultural research. The estimation of pericarp thickness is important to understand the grain quality and storage stability of seeds and can play a crucial role in improving crop yield. In this study, we demonstrate the applicability of fiber-based Bessel beam Fourier domain (FD) optical coherence microscopy (OCM) with a nearly constant high lateral resolution maintained at over ~400 µm for direct non-invasive measurement of the pericarp thickness of two different sorghum genotypes. Whereas measurements based on axial profiles need additional knowledge of the pericarp refractive index, en-face views allow for direct distance measurements. We directly determine pericarp thickness from lateral sections with a 3 µm resolution by taking the width of the signal corresponding to the pericarp at the 1/e threshold. These measurements enable differentiation of the two genotypes with 100% accuracy. We find that trading image resolution for acquisition speed and view size reduces the classification accuracy. Average pericarp thicknesses of 74 µm (thick phenotype) and 43 µm (thin phenotype) are obtained from high-resolution lateral sections, and are in good agreement with previously reported measurements of the same genotypes. Extracting the morphological features of plant seeds using Bessel beam FD-OCM is expected to provide valuable information to the food processing industry and plant breeding programs.

CLICK-17, a DNA enzyme that harnesses ultra-low concentrations of either Cu+ or Cu2+ to catalyze the azide-alkyne 'click' reaction in water.

To enable the optimal, biocompatible and non-destructive application of the highly useful copper (Cu+)-mediated alkyne-azide 'click' cycloaddition in water, we have isolated and characterized a 79-nucleotide DNA enzyme or DNAzyme, 'CLICK-17', that harnesses as low as sub-micromolar Cu+; or, surprisingly, Cu2+ (without added reductants such as ascorbate) to catalyze conjugation between a variety of alkyne and azide substrates, including small molecules, proteins and nucleic acids. CLICK-17's Cu+ catalysis is orders of magnitude faster than that of either Cu+ alone or of Cu+ complexed to PERMUT-17, a sequence-permuted DNA isomer of CLICK-17. With the less toxic Cu2+, CLICK-17 attains rates comparable to Cu+, under conditions where both Cu2+ alone and Cu2+ complexed with a classic accelerating ligand, THPTA, are wholly inactive. Cyclic voltammetry shows that CLICK-17, unlike PERMUT-17, powerfully perturbs the Cu(II)/Cu(I) redox potential. CLICK-17 thus provides a unique, DNA-derived ligand environment for catalytic copper within its active site. As a bona fide Cu2+-driven enzyme, with potential for being evolved to accept only designated substrates, CLICK-17 and future variants promise the fast, safe, and substrate-specific catalysis of 'click' bioconjugations, potentially on the surfaces of living cells.

High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme.

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.

DNA Quadruple Helices in Nanotechnology.

DNA has played an early and powerful role in the development of bottom-up nanotechnologies, not least because of DNA's precise, predictable, and controllable properties of assembly on the nanometer scale. Watson-Crick complementarity has been used to build complex 2D and 3D architectures and design a number of nanometer-scale systems for molecular computing, transport, motors, and biosensing applications. Most of such devices are built with classical B-DNA helices and involve classical A-T/U and G-C base pairs. However, in addition to the above components underlying the iconic double helix, a number of alternative pairing schemes of nucleobases are known. This review focuses on two of these noncanonical classes of DNA helices: G-quadruplexes and the i-motif. The unique properties of these two classes of DNA helix have been utilized toward some remarkable constructions and applications: G-wires; nanostructures such as DNA origami; reconfigurable structures and nanodevices; the formation and utilization of hemin-utilizing DNAzymes, capable of generating varied outputs from biosensing nanostructures; composite nanostructures made up of DNA as well as inorganic materials; and the construction of nanocarriers that show promise for the therapeutics of diseases.

A Long and Reversibly Self-Assembling 1D DNA Nanostructure Built from Triplex and Quadruplex Hybrid Tiles.

We report a new DNA nanostructure, an extended 1-dimensional composite built for the first time out of structurally robust yet conveniently disassembled DNA triple helices, interspersed with short stretches of G-quadruplexes. These "TQ Hybrid" 1-dimensional nanostructures require potassium ions and modestly acidic pH for their formation and are easily disassembled by changes to either of these requirements. We initially prepared and characterized a "monomeric" TQ Hybrid tile; followed by "sticky" TQs tiles, incorporating unique guanine-only sticky ends, that enable efficient self-assembly via G-quartet formation of nanostructures >150 nm in length, as seen with atomic force microscopy and transmission electron microscopy. We anticipate that such DNA TQ Hybrid structures will find unique and varied application as communication modules within larger nanostructures, and as sensors, logic gates, as well as in other aspects of DNA nanotechnology.

Divergent Pair of Ultrasensitive Mechanoelectronic Nanoswitches Made out of DNA.

Mechanoelectronic DNA nanoswitches refer to designed oligonucleotide constructs that are composed of conduction-interrupted duplex stems functionally coupled to ligand recognition motifs; they have been shown to undergo remarkable conduction switching upon binding molecular ligands/analytes. Herein we report a divergent pair of such mechanoelectronic DNA switches, the "signal-on" 3'AA-1 switch and the "signal-off" NB-1 switch, both activated by and responded to mercury ions (Hg2+) at nM levels. We first investigated their charge transport efficiency at a biochemical level, by studying how distinct base sequence at the switches' central three-way junction and at the recognition motif (capable of forming T-Hg2+-T metallo-base pairs) influences their overall conductivity. Gel electrophoresis assays revealed that the presence of two unpaired adenines (AA) at the junction led to "signal-on" behavior with increasing Hg2+ concentration; divergently, absence of these adenines led to a "signal-off" behavior. Upon immobilization on gold electrodes, both DNA switches, with enhanced and inhibited conductivity, respectively, showed excellent sensitivity as well as selectivity toward Hg2+ and can be regenerated for multicycle applications. The high performance of these devices, as both nanoswitches and biosensors with robust and reproducible properties, highlights their potential as an outstanding new class of DNA mechanoelectronic components with built-in biosensing capabilities.

DNA's Encounter with Ultraviolet Light: An Instinct for Self-Preservation?

Photochemical modification is the major class of environmental damage suffered by DNA, the genetic material of all free-living organisms. Photolyases are enzymes that carry out direct photochemical repair (photoreactivation) of covalent pyrimidine dimers formed in DNA from exposure to ultraviolet light. The discovery of catalytic RNAs in the 1980s led to the "RNA world hypothesis", which posits that early in evolution RNA or a similar polymer served both genetic and catalytic functions. Intrigued by the RNA world hypothesis, we set out to test whether a catalytic RNA (or a surrogate, a catalytic DNA) with photolyase activity could be contemplated. In vitro selection from a random-sequence DNA pool yielded two DNA enzymes (DNAzymes): Sero1C, which requires serotonin as an obligate cofactor, and UV1C, which is cofactor-independent and optimally uses light of 300-310 nm wavelength to repair cyclobutane thymine dimers within a gapped DNA substrate. Both Sero1C and UV1C show multiple turnover kinetics, and UV1C repairs its substrate with a quantum yield of ∼0.05, on the same order as the quantum yields of certain classes of photolyase enzymes. Intensive study of UV1C has revealed that its catalytic core consists of a guanine quadruplex (G-quadruplex) positioned proximally to the bound substrate's thymine dimer. We hypothesize that electron transfer from photoexcited guanines within UV1C's G-quadruplex is responsible for substrate photoreactivation, analogous to electron transfer to pyrimidine dimers within a DNA substrate from photoexcited flavin cofactors located within natural photolyase enzymes. Though the analogy to evolution is necessarily limited, a comparison of the properties of UV1C and Sero1C, which arose out of the same in vitro selection experiment, reveals that although the two DNAzymes comparably accelerate the rate of thymine dimer repair, Sero1C has a substantially broader substrate repertoire, as it can repair many more kinds of pyrimidine dimers than UV1C. Therefore, the co-opting of an amino acid-like cofactor by a nucleic acid enzyme in this case contributes functional versatility rather than a greater rate enhancement. In recent work on UV1C, we have succeeded in shifting its action spectrum from the UVB into the blue region of the spectrum and determined that although it catalyzes both repair and de novo formation of thymine dimers, UV1C is primarily a catalyst for thymine dimer repair. Our work on photolyase DNAzymes has stimulated broader questions about whether analogous, purely nucleotide-based photoreactivation also occurs in double-helical DNA, the dominant form of DNA in living cells. Recently, a number of different groups have reported that this kind of repair is indeed operational in DNA duplexes, i.e., that there exist nucleotide sequences that actively protect, by way of photoreactivation (rather than by simply preventing their formation), pyrimidine dimers located proximal to them. Nucleotide-based photoreactivation thus appears to be a salient, if unanticipated, property of DNA and RNA. The phenomenon also offers pointers in the direction of how in primordial evolution-in an RNA world-early nucleic acids may have protected themselves from structural and functional damage wrought by ultraviolet light.

Self-biotinylation of DNA G-quadruplexes via intrinsic peroxidase activity.

The striking and ubiquitous in vitro affinity between hemin and DNA/RNA G-quadruplexes raises the intriguing possibility of its relevance to biology. To date, no satisfactory experimental framework has been reported for investigating such a possibility. Complexation by G-quadruplexes leads to activation of the bound hemin toward catalysis of 1- and 2-electron oxidative reactions, with phenolic compounds being particularly outstanding substrates. We report here a strategy for exploiting that intrinsic peroxidase activity of hemin•G-quadruplex complexes for self-biotinylation of their G-quadruplex component. Such self-biotinylation occurs with good efficiency and high discrimination in vitro, being specific for G-quadruplexes and not for duplexes. The biotinylated DNA, moreover, remains amenable to polymerase chain reaction amplification, rendering it suitable for analysis by ChIP-Seq and related methods. We anticipate that this self-biotinylation methodology will also serve as a sensitive tool, orthogonal to existing ones, for identifying, labeling and pulling down cellular RNA and DNA G-quadruplexes in general, as well as proteins bound to or proximal to such quadruplexes.

Hemin-utilizing G-quadruplex DNAzymes are strongly active in organic co-solvents.

The widespread use of organic solvents in industrial processes has focused in recent years on the utility of "green" solvents - those with less harmful environmental, health, and safety properties - such as methanol and formamide. However, protein enzymes, regarded as green catalysts, are often incompatible with organic solvents. Herein, we have explored the oxidative properties of a Fe(III)-heme, or hemin, utilizing catalytic DNA (heme·DNAzyme) in different green solvent-water mixtures. We find that the peroxidase and peroxygenase activities of the heme·DNAzyme are strongly enhanced in 20-30% v/v methanol or formamide, relative to water alone. Protic solvent content of >30% v/v gradually diminishes heme·DNAzyme catalytic activity; however, the heme·DNAzyme is still active in as high as 80% v/v methanol. In contrast to protic solvents, aqueous dimethylformamide solutions largely inhibit heme·DNAzyme activity. In view of the strong catalytic activity of heme·DNAzyme in aqueous methanol, we were able to determine that a 60% v/v methanol-water mixture gives the most optimal yield of the dibenzothiophene sulfoxide (DBTO) oxidation product of petroleum-derived dibenzothiophene (DBT). The high product yield reflects both DNAzyme catalysis and a high substrate availability. Overall, these results emphasize the excellent promise of G-quadruplex forming DNA catalysts in application to "greener" industrial chemistry. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

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.

DNA Repair by DNA: The UV1C DNAzyme Catalyzes Photoreactivation of Cyclobutane Thymine Dimers in DNA More Effectively than Their de Novo Formation.

UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA.

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.

DNA mechatronic devices switched by K⁺ and by Sr²âº 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 Sr(2+), 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 Sr(2+)-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.

Functional DNA switches: rational design and electrochemical signaling.

Recent developments in nanoscience research have demonstrated that DNA switches (rationally designed DNA nanostructures) constitute a class of versatile building blocks for the fabrication and assembly of electronic devices and sensors at the nanoscale. Functional DNA sequences and structures such as aptamers, DNAzymes, G-quadruplexes, and i-motifs can be readily prepared in vitro, and subsequently adapted to an electrochemical platform by coupling with redox reporters. The conformational or conduction switching of such electrode-bound DNA modules in response to an external stimulus can then be monitored by conventional voltammetric measurements. In this review, we describe how we are able to design and examine functional DNA switches, particularly those systems that utilize electrochemical signaling. We also discuss different available options for labeling functional DNA with redox reporters, and comment on the function-oriented signaling pathways.

Assessment of Vitamin D status In Patients of Chronic Low Back Pain of Unknown Etiology.

Low back pain is very disabling and dispiriting because of the physical impediment it causes and its psychological effects. Innumerable factors have been implicated in its etiology. In spite of improvements in diagnostic modalities, a considerable number of such cases fall in the ambiguous zone of unknown etiology or 'idiopathic.'Early diagnosis of low back pain will allow effective prevention and treatment to be offered. This study was conducted to assess the contribution of vitamin D levels and other biochemical factors to chronic low back pain in such cases. All patients attending the orthopedics OPD for low back pain in whom a precise anatomical cause could not be localized, were prospectively enrolled in this study. We measured serum levels of glucose, calcium, phosphorus, uric acid, rheumatoid factor, C reactive protein, alkaline phosphatase, total protein, albumin and 25 (OH) D concentrations in 200 cases and 200 control samples. The patients showed significantly lower vitamin D levels compared to controls with p value < 0.0001. The maximum number of low back pain patients were in the age group of 31-50 years (42 %).The average BMI was 23.27 ± 5.17 kg/sq m, 73 % of total patient population were females and 27 % were known case of type 2 diabetes mellitus. Calcium, alkaline phosphatase, was positively correlated with vitamin D and glucose showed a negative correlation with vitamin D in the patient population. The problem of low back pain provides a challenge to health care providers. The problem in developing countries is compounded by ignorance to report for early treatment and occupational compulsions in rural areas and sedentary lifestyle in urban youth. The authors strongly recommend early frequent screening for vitamin D along with glucose, protein, albumin, calcium, phosphorus, CRP as part of general health checkup for non-specific body pain, especially low back pain.

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.