Covalently linked DNA nanotubes.

The present study introduces an approach to prepare covalently linked DNA nanotubes. A circular DNA that includes at its opposite poles thiol and amine functionalities acts as the building block for the construction of the DNA nanotubes. The circular DNA is cross-linked with a bis-amide-modified nucleic acid to yield DNA nanowires, and these are subsequently cross-linked by a bis-thiolated nucleic acid to yield the DNA nanotubes. Alternatively, a circular DNA that includes four amine functionalities on its poles is cross-linked in one-step by the bis-thiolated nucleic acid to yield the nanotubes. The resulting nanostructures are stable and nonseparable upon heating.

Cooperative multicomponent self-assembly of nucleic acid structures for the activation of DNAzyme cascades: a paradigm for DNA sensors and aptasensors.

The activation of a DNAzyme cascade by the cooperative self-assembly of multicomponent nucleic acid structures is suggested as a method for the amplified sensing of DNA, or the specific substrates of aptamers. According to one configuration, the DNA analyte 1 is detected by two tailored nucleic acids 2 and 3 that form a multicomponent supramolecular structure with a ribonucleobase-containing quasi-circular DNA 4, but only upon the concomitant hybridization with 1. The resulting supramolecular nucleic acid structure includes the Mg(2+)-dependent DNAzyme that cleaves the ribonucleobase site of 4. The cleavage of the quasi-circular DNA 4 results in the fragmentation of the supramolecular structure and the release of two horseradish peroxidase (HRP) mimicking units that were incorporated in the blocked quasi-circular DNA 4. The HRP-mimicking DNAzyme catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS(2-)) by H(2)O(2) to ABTS(*-), and the product provided the colorimetric readout signal for the analyzed DNA. The method enabled the analysis of DNA with a detection limit of 1 x 10(-12) M. Similarly, an analogous DNAzyme cascade was activated by the low-molecular-weight substrates, adenosine triphosphate (ATP) or cocaine. This was induced by the self-assembly of nucleic acids that included fragments of the respective aptamers and the Mg(2+)-dependent DNAzyme. Furthermore, nucleic acids consisting of fragments of the aptamers against ATP or cocaine and fragments of the HRP-mimicking DNAzyme self-assemble, in the presence of the respective substrates, to the active DNAzyme structure that catalyzes the oxidation of ABTS(2-) by H(2)O(2) to form the colored product ABTS(*-). The resulting product provided the readout signal for the recognition events. The cooperative interaction in the formation of the supramolecular nucleic acid assemblies and the activation of the DNAzymes are discussed.

Parallel analysis of two analytes in solutions or on surfaces by using a bifunctional aptamer: applications for biosensing and logic gate operations.

A bifunctional aptamer that includes two aptamer units for cocaine and adenosine 5'-monophosphate (AMP) is blocked by a nucleic acid to form a hybrid structure with two duplex regions. The blocked bifunctional aptamer assembly is used as a functional structure for the simultaneous sensing of cocaine or AMP. The blocked bifunctional aptamer is dissociated by either of the two analytes, and the readout of the separation of the sensing structure is accomplished by a colorimetric detection, by a released DNAzyme, or by electronic means that use Faradaic impedance spectroscopy or field-effect transistors. In one configuration, the blocked bifunctional aptamer structure is separated by the substrates cocaine or AMP, and the displaced blocker units act as a horseradish peroxidase-mimicking DNAzyme that permits the colorimetric detection of the analytes. In the second system, the blocked bifunctional aptamer hybrid is associated with a Au electrode. The displacement of the aptamer by any of the substrates alters the interfacial electron transfer resistance at the electrode surface, thus providing an electronic signal for the sensing process. In the third configuration, the blocked aptamer hybrid is linked to the gate of a field-effect transistor device. The separation of the complex by means of any of the analytes, cocaine, or AMP alters the gate potential, and this allows the electronic transduction of the sensing process by following the changes in the gate-to-source potentials. The different systems enable not only the simultaneous detection of the two analytes, but they provide a functional assembly that performs a logic gate "OR" operation.

A DNAzyme cascade for the amplified detection of Pb(2+) ions or L-histidine.

DNAzyme cascades activated by Pb(2+)- or L-histidine-dependent DNAzymes yield the horseradish peroxidase-mimicking catalytic nucleic acids that enable the colorimetric or chemiluminescence detection of Pb(2+) or L-histidine.

Proteins modified with DNAzymes or aptamers act as biosensors or biosensor labels.

Hybrid systems composed of a glucose oxidase (GOx)/peroxidase-mimicking DNAzyme, and of microperoxidase-11 (MP-11)/anti-thrombin aptamer were synthesized. The hybrid systems were employed as amplifying labels for the colorimetric or chemiluminescence detection of an enzyme functions, and thrombin analysis, respectively. In the GOx/DNAzyme system, the GOx-mediated oxidation of glucose led to the formation of H(2)O(2), and this activated the oxidation of ABTS to a colored product, or to the generation of chemiluminescence in the presence of luminol. The MP-11/anti-thrombin aptamer enabled the amplified analysis of thrombin by the MP-11-mediated generation of chemiluminescence in the presence of luminol/H(2)O(2).

Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold.

DNAzymes for sensing, nanobiotechnology and logic gate applications.

Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, "DNA machines" that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.

Fluorescence detection of DNA by the catalytic activation of an aptamer/thrombin complex.

A conjugate consisting of a thrombin aptamer tethered to the thrombin, Th, with a sensing nucleic acid (1) is used for the optical detection of DNA. The thrombin/aptamer complex blocks the biocatalytic functions of Th. Hybridization of the analyte DNA (2) to the sensing nucleic acid 1 yields a rigid duplex that detaches the aptamer from Th, a process that activates the protein toward the hydrolysis of bis(p-tosyl-Gly-Pro-Arg)-R110 (3) to the rhodamine 110 fluorophore (4). The system allows the DNA sensing with a sensitivity limit of 1 x 10-8 M. The aptamer/Th conjugate is also immobilized on glass slides for the optical detection of DNA. The dissociation of the aptamer/Th complex upon hybridization and the subsequent dehybridization of the duplex and the regeneration of the catalytically inactive Th/aptamer complex duplicate machinery functions.

An Os(II)--bisbipyridine--4-picolinic acid complex mediates the biocatalytic growth of au nanoparticles: optical detection of glucose and acetylcholine esterase inhibition.

The complex Os(II)-bisbipyridine-4-picolinic acid, [Os(bpy)(2)PyCO(2)H](2+) (1), mediates the biocatalyzed growth of Au nanoparticles, Au NPs, and enables the spectroscopic assay of biocatalyzed transformations and enzyme inhibition by following the Au NP plasmon absorbance. In one system, [Os(bpy)(2)PyCO(2)H](2+) mediates the biocatalyzed oxidation of glucose and the growth of Au NPs in the presence of glucose oxidase, GOx, AuCl(4) (-), citrate and Au NP seeds. The mechanism of the Au NPs growth involves the oxidation of the [Os(bpy)(2)PyCO(2)H](2+) complex by AuCl(4) (-) to form [Os(bpy)(2)PyCO(2)H](3+) and Au(I). The [Os(bpy)(2)PyCO(2)H](3+) complex mediates the GOx biocatalyzed oxidation of glucose and the regeneration of the mediator 1. Citrate reduces Au(I) and enlarges the Au seeds by the catalytic deposition of gold on the Au NP seeds. In the second system, the enzyme acetylcholine esterase, AChE, is assayed by the catalytic growth of the Au NPs. The hydrolysis of acetylcholine (2) by AChE to choline is followed by the [Os(bpy)(2)PyCO(2)H](3+) mediated oxidation of choline to betaine and the concomitant growth of the Au NPs. The mediated growth of the Au NPs is inhibited by 1,5-bis(4-allyldimethylammonium-phenyl)pentane-3-one dibromide (3). A competitive inhibition process was demonstrated (K(M)=0.13 mM, K(I)=2.6 microM) by following the growth of the Au NPs.

Shape and color of au nanoparticles follow biocatalytic processes.

The NAD(P)H-mediated growth of Au nanoparticles (NPs) in the presence of ascorbic acid, AuCl4-, and cetyltrimethylammonium bromide leads to the formation of shaped NP structures consisting of dipods, tripods, and tetrapods. The shaped particles exhibit a red-shifted plasmon absorbance at lambda = 680 nm, consistent with the existence of a longitudinal plasmon exciton. High-resolution transmission electron microscopy analysis of the tripod and tetrapod structures reveals directional growth along the <211> and <010> directions, respectively. The shaped Au NPs could be generated by a biocatalytic process using alcohol dehydrogenase, NAD+, and ethanol, and the resulting blue color provides a colorimetric test for ethanol.

Aptamer-functionalized Au nanoparticles for the amplified optical detection of thrombin.

The catalytic enlargement of aptamer-functionalized Au nanoparticles amplifies the optical detection of aptamer-thrombin complexes in solution and on surfaces.