Selective binding of c-MYC G-quadruplex caged in a dsDNA by a hemopeptide.

The microperoxidase-11 hemopeptide exhibits configuration-dependent selectivity for guanine-quadruplexes by specifically uncaging c-MYC guanine-quadruplexes from a duplex DNA.

Redox- and metal-directed structural diversification in designed metalloprotein assemblies.

Herein we describe a designed protein building block whose self-assembly behaviour is dually gated by the redox state of disulphide bonds and the identity of exogenous metal ions. This protein construct is shown - through extensive structural and biophysical characterization - to access five distinct oligomeric states, exemplifying how the complex interplay between hydrophobic, metal-ligand, and reversible covalent interactions could be harnessed to obtain multiple, responsive protein architectures from a single building block.

Design of metal-mediated protein assemblies via hydroxamic acid functionalities.

The self-assembly of proteins into sophisticated multicomponent assemblies is a hallmark of all living systems and has spawned extensive efforts in the construction of novel synthetic protein architectures with emergent functional properties. Protein assemblies in nature are formed via selective association of multiple protein surfaces through intricate noncovalent protein-protein interactions, a challenging task to accurately replicate in the de novo design of multiprotein systems. In this protocol, we describe the application of metal-coordinating hydroxamate (HA) motifs to direct the metal-mediated assembly of polyhedral protein architectures and 3D crystalline protein-metal-organic frameworks (protein-MOFs). This strategy has been implemented using an asymmetric cytochrome cb562 monomer through selective, concurrent association of Fe3+ and Zn2+ ions to form polyhedral cages. Furthermore, the use of ditopic HA linkers as bridging ligands with metal-binding protein nodes has allowed the construction of crystalline 3D protein-MOF lattices. The protocol is divided into two major sections: (1) the development of a Cys-reactive HA molecule for protein derivatization and self-assembly of protein-HA conjugates into polyhedral cages and (2) the synthesis of ditopic HA bridging ligands for the construction of ferritin-based protein-MOFs using symmetric metal-binding protein nodes. Protein cages are analyzed using analytical ultracentrifugation, transmission electron microscopy and single-crystal X-ray diffraction techniques. HA-mediated protein-MOFs are formed in sitting-drop vapor diffusion crystallization trays and are probed via single-crystal X-ray diffraction and multi-crystal small-angle X-ray scattering measurements. Ligand synthesis, construction of HA-mediated assemblies, and post-assembly analysis as described in this protocol can be performed by a graduate-level researcher within 6 weeks.

Constructing protein polyhedra via orthogonal chemical interactions.

Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6-a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, 'one-pot' coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe3+ and Zn2+ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures7,8 and are, to our knowledge, unique among designed systems9-13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design.

A Bis-Zn2+ -Pyridyl-Salen-Type Complex Conjugated to the ATP Aptamer: An ATPase-Mimicking Nucleoapzyme.

Catalytic nucleic acids consisting of a bis-Zn2+ -pyridyl-salen-type ([di-ZnII 3,5 bis(pyridinylimino) benzoic acid]) complex conjugated to the ATP aptamer act as ATPase-mimicking catalysts (nucleoapzymes). Direct linking of the Zn2+ complex to the 3'- or 5'-end of the aptamer (nucleoapzymes I and II) or its conjugation to the 3'- or 5'-end of the aptamer through bis-thymidine spacers (nucleoapzymes III and IV) provided a set of nucleoapzymes exhibiting variable catalytic activities. Whereas the separated bis-Zn2+ -pyridyl-salen-type catalyst and the ATP aptamer do not show any noticeable catalytic activity, the 3'-catalyst-modified nucleoapzyme (nucleoapzyme IV) and, specifically, the nucleoapzyme consisting of the catalyst linked to the 3'-position through the spacer (nucleoapzyme III) reveal enhanced catalytic features in relation to the analogous nucleoapzyme substituted at the 5'-position (kcat =4.37 and 6.88 min-1 , respectively). Evaluation of the binding properties of ATP to the different nucleoapzyme and complementary molecular dynamics simulations suggest that the distance separating the active site from the substrate linked to the aptamer binding site controls the catalytic activities of the different nucleoapzymes.

Supramolecular micelle-based nucleoapzymes for the catalytic oxidation of dopamine to aminochrome.

Lipidated DNAzymes or a lipidated Cu(II)-complex and lipidated aptamer sequences form supramolecular assemblies of micellar nucleoapzymes for the enhanced oxidation of dopamine to aminochrome. The catalytic functions of the micellar nucleoapzymes are attributed to the concentration of the substrate, using the aptamer units, in close proximity to the active sites.

Nucleoapzymes: Hemin/G-Quadruplex DNAzyme-Aptamer Binding Site Conjugates with Superior Enzyme-like Catalytic Functions.

A novel concept to improve the catalytic functions of nucleic acids (DNAzymes) is introduced. The method involves the conjugation of a DNA recognition sequence (aptamer) to the catalytic DNAzyme, yielding a hybrid structure termed "nucleoapzyme". Concentrating the substrate within the "nucleoapzyme" leads to enhanced catalytic activity, displaying saturation kinetics. Different conjugation modes of the aptamer/DNAzyme units and the availability of different aptamer sequences for a substrate provide diverse means to design improved catalysts. This is exemplified with (i) The H2O2-mediated oxidation of dopamine to aminochrome using a series of hemin/G-quadruplex-dopamine aptamer nucleoapzymes. All nucleoapzymes reveal enhanced catalytic activities as compared to the separated DNAzyme/aptamer units, and the most active nucleoapzyme reveals a 20-fold enhanced activity. Molecular dynamics simulations provide rational assessment of the activity of the various nucleoapzymes. The hemin/G-quadruplex-aptamer nucleoapzyme also stimulates the chiroselective oxidation of L- vs D-DOPA by H2O2. (ii) The H2O2-mediated oxidation of N-hydroxy-L-arginine to L-citrulline by a series of hemin/G-quadruplex-arginine aptamer conjugated nucleoapzymes.

Rational design of supramolecular hemin/G-quadruplex-dopamine aptamer nucleoapzyme systems with superior catalytic performance.

The rational design of a set of hemin/G-quadruplex (hGQ)-dopamine binding aptamer (DBA) conjugates, acting as nucleoapzymes, is described. The nucleoapzyme constructs consist of a hGQ DNAzyme as a catalytic unit and DBA as a substrate binding unit that are brought into spatial proximity by a duplex scaffold composed of complementary oligonucleotide strands. When the hGQ unit is linked to the duplex scaffold via a single-strand DNA tether of variable length, the resulting nucleoapzymes reveal a moderate catalytic enhancement toward the H2O2-mediated oxidation of dopamine to aminochrome as compared to the process stimulated by the separated hGQ and DBA units (5-7 fold enhancement). This limited enhancement is attributed to inappropriate spatial positioning of the hGQ in respect to the dopamine binding site, and/or to the flexibility of the tether that links the hGQ catalytic site to the double-stranded scaffold. To solve this, rigidification of the hGQ/DBA conjugates by triplex oligonucleotide structures that anchor the hGQ to a duplex domain associated with the DBA units was achieved. By the sequential, programmed, triplex-controlled rigidification of the hGQ/DBA structure, a nucleoapzyme with superior catalytic activity toward the oxidation of dopamine to aminochrome is identified (30-fold catalytic enhancement). Molecular dynamics simulations reveal that in the resulting highly active rigidified nucleoapzyme structure, the hGQ catalytic site is positioned in spatial proximity to the opening of the DBA substrate binding site, thus rationalizing and supporting the enhanced catalytic functions of the system. Finally, the most active nucleoapzyme system was subjected to fuel- and anti-fuel strands that separate and re-assemble the nucleoapzyme structure, allowing "ON" and "OFF" switching of the nucleoapzyme catalytic functions.

G-Quadruplex-Stimulated Optical and Electrocatalytic DNA Switches.

The K(+) /18-crown-6-(or [2.2.2] cryptand)-stimulated formation and dissociation of G-quadruplex nanostructures lead to the cyclic and switchable photonic and electrocatalytic molecular devices.

Computational docking simulations of a DNA-aptamer for argininamide and related ligands.

The binding properties of sequence-specific nucleic acids (aptamers) to low-molecular-weight ligands, macromolecules and even cells attract substantial scientific interest. These ligand-DNA complexes found different applications for sensing, nanomedicine, and DNA nanotechnology. Structural information on the aptamer-ligand complexes is, however, scarce, even though it would open-up the possibilities to design novel features in the complexes. In the present study we apply molecular docking simulations to probe the features of an experimentally documented L-argininamide aptamer complex. The docking simulations were performed using AutoDock 4.0 and YASARA Structure software, a well-suited program for following intermolecular interactions and structures of biomolecules, including DNA. We explored the binding features of a DNA aptamer to L-argininamide and to a series of arginine derivatives or arginine-like ligands. We find that the best docking results are obtained after an energy-minimization of the parent ligand-aptamer complexes. The calculated binding energies of all mono-substituted guanidine-containing ligands show a good correlation with the experimentally determined binding constants. The results provide valuable guidelines for the application of docking simulations for the prediction of aptamer-ligand structures, and for the design of novel features of ligand-aptamer complexes.

Addressing, amplifying and switching DNAzyme functions by electrochemically-triggered release of metal ions.

The design of artificial cells, which mimic the functions of native cells, is an ongoing scientific goal. The development of stimuli-responsive chemical systems that stimulate cascaded catalytic transformations, trigger chemical networks, and control vectorial branched transformations and dose-controlled processes, are the minimum requirements for mimicking cell functions. We have studied the electrochemical programmed release of ions from electrodes, which trigger selective DNAzyme-driven chemical reactions, cascaded reactions that self-assemble catalytic DNAzyme polymers, and the ON-OFF switching and dose-controlled operation of catalytic reactions. The addressable and potential-controlled release of Pb2+ or Ag+ ions into an electrolyte that includes a mixture of nucleic acids, results in the metal ion-guided selection of nucleic acids yielding the formation of specific DNAzymes, which stimulate orthogonal reactions or activate DNAzyme cascades.

Switchable enzyme/DNAzyme cascades by the reconfiguration of DNA nanostructures.

Mimicking cellular transformations and signal transduction pathways by means of biocatalytic cascades proceeding in organized media is a scientific challenge. We describe two DNA machines that enable the "ON/OFF" switchable activation and deactivation of three-component biocatalytic cascades. One system consists of a reconfigurable DNA tweezers-type structure, whereas in the second system the catalytic cascade proceeds on a switchable DNA clamp scaffold. The three-component catalytic cascades consist of ß-galactosidase (ß-Gal), glucose oxidase (GOx), and the K(+) -ion-stabilized hemin-G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme. The hemin-G-quadruplex-bridged closed structure of the tweezers or clamp allows the biocatalytic cascades to operate (switched "ON''), whereas separation of the hemin-G-quadruplex by means of 18-crown-6-ether opens the tweezers/clamp structures, thus blocking the catalytic cascade (switched "OFF"). This study is complemented by two-component, switchable biocatalytic cascades composed of GOx and hemin-G-quadruplex assembled on hairpin-bridged DNA tweezers or clamp nanostructures.

Ag nanocluster/DNA hybrids: functional modules for the detection of nitroaromatic and RDX explosives.

Luminescent Ag nanoclusters (NCs) stabilized by nucleic acids are implemented as optical labels for the detection of the explosives picric acid, trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The sensing modules consist of two parts, a nucleic acid with the nucleic acid-stabilized Ag NCs and a nucleic acid functionalized with electron-donating units, including L-DOPA, L-tyrosine and 6-hydroxy-L-DOPA, self-assembled on a nucleic acid scaffold. The formation of donor-acceptor complexes between the nitro-substituted explosives, exhibiting electron-acceptor properties, and the electron-donating sites, associated with the sensing modules, concentrates the explosives in close proximity to the Ag NCs. This leads to the electron-transfer quenching of the luminescence of the Ag NCs by the explosive molecule. The quenching of the luminescence of the Ag NCs provides a readout signal for the sensing process. The sensitivities of the analytical platforms are controlled by the electron-donating properties of the donor substituents, and 6-hydroxy-L-DOPA was found to be the most sensitive donor. Picric acid, TNT, and RDX are analyzed with detection limits corresponding to 5.2 × 10(-12) M, 1.0 × 10(-12) M, and 3.0 × 10(-12) M, respectively, using the 6-hydroxy-L-DOPA-modified Ag NCs sensing module.

DNA sensors and aptasensors based on the hemin/G-quadruplex-controlled aggregation of Au NPs in the presence of L-cysteine.

L-cysteine induces the aggregation of Au nanoparticles (NPs), resulting in a color transition from red to blue due to interparticle plasmonic coupling in the aggregated structure. The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme catalyzes the aerobic oxidation of L-cysteine to cystine, a process that inhibits the aggregation of the NPs. The degree of inhibition of the aggregation process is controlled by the concentration of the DNAzyme in the system. These functions are implemented to develop sensing platforms for the detection of a target DNA, for the analysis of aptamer-substrate complexes, and for the analysis of L-cysteine in human urine samples. A hairpin DNA structure that includes a recognition site for the DNA analyte and a caged G-quadruplex sequence, is opened in the presence of the target DNA. The resulting self-assembled hemin/G-quadruplex acts as catalyst that controls the aggregation of the Au NPs. Also, the thrombin-binding aptamer folds into a G-quadruplex nanostructure upon binding to thrombin. The association of hemin to the resulting G-quadruplex aptamer-thrombin complex leads to a catalytic label that controls the L-cysteine-mediated aggregation of the Au NPs. The hemin/G-qaudruplex-controlled aggregation of Au NPs process is further implemented for visual and spectroscopic detection of L-cysteine concentration in urine samples.

Analysis of telomerase by the telomeric hemin/G-quadruplex-controlled aggregation of au nanoparticles in the presence of cysteine.

Telomeres are guanosine-rich nucleic-acid chains that fold, in the presence of K(+) ions and hemin, into the telomeric hemin/G-quadruplex structure, exhibiting horseradish peroxidase mimicking functions. The telomeric hemin/G-quadruplex structures catalyze the oxidation of thiols (e.g., l-cysteine) into disulfides (e.g., cystine). As l-cysteine stimulates the aggregation of Au nanoparticles (NPs), accompanied by absorbance changes from red (individual Au NPs) to blue (aggregated Au NPs), the process is implemented to quantitatively analyze the activity (content) of telomerase, a versatile biomarker for cancer cells. Telomerase extracted from 293T cancer cells catalyzes, in the presence of a dNTPs mixture and an appropriate primer probe, the telomerization process, leading to the generation of catalytic telomeric hemin/G-quadruplex chains that control the l-cysteine-mediated aggregation of Au NPs. The extent of aggregation is thus controlled by the concentration of telomerase. The method enabled the detection of telomerase with a detection limit of 27 cells/µL. The spectral changes accompanying the aggregation of Au NPs are further supported by transmission electron microscopy imaging.

Hemin/G-quadruplex-catalyzed aerobic oxidation of thiols to disulfides: application of the process for the development of sensors and aptasensors and for probing acetylcholine esterase activity.

This study describes the novel hemin/G-quadruplex DNAzyme-catalyzed aerobic oxidation of thiols to disulfides and the respective mechanism. The mechanism of the reaction involves the DNAzyme-catalyzed oxidation of thiols to disulfides and the thiol-mediated autocatalytic generation of H2O2 from oxygen. The coupling of a concomitant H2O2-mediated hemin/G-quadruplex-catalyzed oxidation of Amplex Red to the fluorescent resorufin as a transduction module provides a fluorescent signal for probing the catalyzed oxidation of the thiol to disulfides and for probing sensing processes that yield the hemin/G-quadruplex as a functional label. Accordingly, a versatile sensing method for analyzing thiols (L-cysteine, glutathione) using the H2O2-mediated DNAzyme-catalyzed oxidation of Amplex Red to the resorufin was developed. Also, the L-cysteine and Amplex Red system was implemented as an auxiliary fluorescent transduction module for probing recognition events that form the catalytic hemin/G-quadruplex structures. This is exemplified with the development of thrombin aptasensor. The thrombin/thrombin binding aptamer recognition complex binds hemin, and the resulting catalytic complex activates the auxiliary transduction module, involving the aerobic oxidation of l-cysteine and the concomitant formation of the fluorescent resorufin. Finally, the hemin/G-quadruplex DNAzyme/Amplex Red system was used to follow the activity of acetylcholine esterase, AChE, and to probe its inhibition. The AChE-catalyzed hydrolysis of acetylthiocholine to the thiol-functionalized thiocholine enabled the probing of the enzymatic activity of AChE through the hemin/G-quadruplex-catalyzed aerobic oxidation of thiocholine to the respective disulfide and the concomitant generation of the fluorescent resorufin product.

Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase.

Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition.

Chemiluminescence and chemiluminescence resonance energy transfer (CRET) aptamer sensors using catalytic hemin/G-quadruplexes.

The incorporation of hemin into the thrombin/G-quadruplex aptamer assembly or into the ATP/G-quadruplex nanostructure yields active DNAzymes that catalyze the generation of chemiluminescence. These catalytic processes enable the detection of thrombin and ATP with detection limits corresponding to 200 pM and 10 µM, respectively. The conjugation of the antithrombin or anti-ATP aptamers to CdSe/ZnS semiconductor quantum dots (QDs) allowed the detection of thrombin or ATP through the luminescence of the QDs that is powered by a chemiluminescence resonance energy-transfer (CRET) process stimulated by the hemin/G-quadruplex/thrombin complex or the hemin/G-quadruplex/ATP nanostructure, in the presence of luminol/H(2)O(2). The advantages of applying the CRET process for the detection of thrombin or ATP, by the resulting hemin/G-quadruplex DNAzyme structures, are reflected by low background signals and the possibility to develop multiplexed aptasensor assays using different sized QDs.

Electrochemical, photoelectrochemical, and surface plasmon resonance detection of cocaine using supramolecular aptamer complexes and metallic or semiconductor nanoparticles.

Metallic or semiconductor nanoparticles (NPs) are used as labels for the electrochemical, photoelectrochemical, or surface plasmon resonance (SPR) detection of cocaine using a common aptasensor configuration. The aptasensors are based on the use of two anticocaine aptamer subunits, where one subunit is assembled on a Au support, acting as an electrode or a SPR-active surface, and the second aptamer subunit is labeled with Pt-NPs, CdS-NPs, or Au-NPs. In the different aptasensor configurations, the addition of cocaine results in the formation of supramolecular complexes between the NPs-labeled aptamer subunits and cocaine on the metallic surface, allowing the quantitative analysis of cocaine. The supramolecular Pt-NPs-aptamer subunits-cocaine complex allows the detection of cocaine by the electrocatalyzed reduction of H(2)O(2). The photocurrents generated by the CdS-NPs-labeled aptamer subunits-cocaine complex, in the presence of triethanol amine as a hole scavenger, allows the photoelectrochemical detection of cocaine. The supramolecular Au-NPs-aptamer subunits-cocaine complex generated on the Au support allows the SPR detection of cocaine through the reflectance changes stimulated by the electronic coupling between the localized plasmon of the Au-NPs and the surface plasmon wave. All aptasensor configurations enable the analysis of cocaine with a detection limit in the range of 10(-6) to 10(-5) M. The major advantage of the sensing platform is the lack of background interfering signals.