Nucleic-Acid Structures as Intracellular Probes for Live Cells.

The chemical composition of cells at the molecular level determines their growth, differentiation, structure, and function. Probing this composition is powerful because it provides invaluable insight into chemical processes inside cells and in certain cases allows disease diagnosis based on molecular profiles. However, many techniques analyze fixed cells or lysates of bulk populations, in which information about dynamics and cellular heterogeneity is lost. Recently, nucleic-acid-based probes have emerged as a promising platform for the detection of a wide variety of intracellular analytes in live cells with single-cell resolution. Recent advances in this field are described and common strategies for probe design, types of targets that can be identified, current limitations, and future directions are discussed.

Dynamic sandwich-type electrochemical assay for protein quantification and protein-protein interaction.

The study of protein-protein interactions (PPIs) plays an important role in the understanding of biological systems; however, the established methods for PPI analysis often involve cumbersome sample preparation, multiple detecting steps, and costly instruments. Here we report a versatile and sensitive electrochemical method based on PPI-induced distinctive migration behavior of DNA deoxyribozyme (DNAzyme) on an electrode surface. In this method, the cleavage activity of DNAzyme toward the substrate DNA modified on the electrode surface is inversely correlated with the hydrodynamic diameter of the macromolecule attached to it. By making full use of this principle in an inexpensive electrochemical format that is named the dynamic sandwich-type electrochemical assay (dSTEA), we can probe into the presence of large macromolecules in a single-step procedure. Moreover, we can not only detect sub-picomolar protein interaction events but also analyze the assembly of kinase in the whole cell extract. This novel signaling mechanism proposed in this work may broaden the applicability of DNAzyme-based electrochemical assays and it may also have great potential for applications in other interfacial sensor developments.

Microfluidic bead trap as a visual bar for quantitative detection of oligonucleotides.

We demonstrate a microfluidic bead trap capable of forming a dipstick-type bar visible to the naked eye for simple and quantitative detection of oligonucleotides. We use magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are connected and form MMPs-targets-PMPs when target oligonucleotides are present, leaving free PMPs with a number inversely proportional to the amount of targets. Using a capillary flow-driven microfluidic circuitry consisting of a magnetic separator to remove the MMPs-targets-PMPs, the free PMPs can be trapped at the narrowing nozzle downstream, forming a visual bar quantifiable based on the length of PMP accumulation. Such a power-free and instrument-free platform enables a limit of detection at 13 fmol (0.65 nM in 20 µl, S/N = 3) of oligonucleotides and is compatible with single-nucleotide polymorphisms and operation in a complex bio-fluid. Moreover, using DNAzyme as the target oligonucleotide that catalyzes a specific hydrolytic cleavage in the presence of lead ions, we demonstrate a model application that detects lead ions with a limit of detection of 12.2 nM (2.5 µg l-1), providing quantitative and visual detection of lead contamination at resource-limited sites.

Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation.

Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy.

Nanoparticles-free fluorescence anisotropy amplification assay for detection of RNA nucleotide-cleaving DNAzyme activity.

Fluorescence anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large fluorescence anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced fluorescence anisotropy assay for detection of DNAzyme activity. A Pb(2+)-dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5'-end with the DNAzyme strand containing an extended three G bases at the 3'-end. By this design, we demonstrate that both fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb(2+)-induced large fluorescence anisotropy change (|Δr| = 0.168). The limit of detection for Pb(2+) is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we show an extended assay for evaluation of the interactions of Pb(2+) with cysteine and glutathione by the detection of GR5 DNAzyme activity. Collectively, we developed a novel fluorescence anisotropy amplification assay, enabling us to detect DNAzyme activity and associated cofactors and inhibitors and to characterize the Pb(2+)-chelation capability of free thiols.

A new colorimetric strategy for monitoring caspase 3 activity by HRP-mimicking DNAzyme-peptide conjugates.

A new method for caspase 3 activity assay has been developed based on HRP-mimicking DNAzyme-peptide conjugates. The mechanism of detection was based on the specific cleavage of DEVD-peptides by active caspase 3 for recognition and the catalytic properties of HRP-mimicking DNAzymes for signal amplification. Under optimal conditions, the detection limit of caspase 3 was 0.89 nM. The proposed method was also successfully applied for the detection of caspase 3 in apoptosis cell lysates.

Tetra(p-tolyl)borate-functionalized solvent polymeric membrane: a facile and sensitive sensing platform for peroxidase and peroxidase mimetics.

The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer.

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

A novel electrochemical aptasensor for thrombin detection based on the hybridization chain reaction with hemin/G-quadruplex DNAzyme-signal amplification.

In this work, a novel signal amplification electrochemical aptasensor for the sensitive and selective detection of thrombin was successfully fabricated. The amplification method was based on the hybridization chain reaction (HCR) and a pseudobienzyme electrocatalytic system. HCR-based double-stranded DNA (dsDNA) polymers not only constructed an effective carrier for anchoring larger amounts of electron mediator methylene blue (MB) into the DNA duplexes to produce a strong differential pulse voltammetry (DPV) signal, but also resulted in the formation of hemin/G-quadruplex DNAzymes nanowires by intercalating hemin into two induced single-stranded DNA (ssDNA). With the addition of NADH into the electrolytic cell, the hemin/G-quadruplex acting as an NADH oxidase and HRP-mimicking DNAzyme for the pseudobienzyme amplifying system could in situ biocatalyze the formation of H2O2 with local concentrations and low transfer loss resulting in dramatic signal enhancements. The binding event can be detected by a decrease in the integrated charge of MB which electrostatically absorbed onto dsDNA polymers. In the presence of thrombin, the dsDNA polymers associated with MB and hemin/G-quadruplex structures were removed from the electrode surface, leading to a significant decrease of redox current. DPV signals of MB provided quantitative measures of the concentrations of thrombin, with a linear calibration range of 0.01-50 nM and a detection limit of 2 pM. Moreover, the resulting aptasensor also exhibited good specificity, acceptable reproducibility and stability, indicating that the present strategy was promising for broad potential application in clinic assay and various protein analyses.

High-throughput quantitative screening of peroxidase-mimicking DNAzymes on a microarray by using electrochemical detection.

Some guanine-rich DNA sequences, which are called DNAzymes, can adopt G-quadruplex structures and exhibit peroxidase activity by binding with hemin. Although known DNAzymes show less activity than horseradish peroxidase, they have the potential to be widely used for the detection of target molecules in enzyme-linked immunosorbent assays if sequences that exhibit higher activity can be identified. However, techniques for achieving this have not yet been described. Therefore, we compared the DNAzyme activities of more than 1000 novelistically designed sequences with that of the original DNAzyme by using an electrochemical detection system on a 12K DNA microarray platform. To the best of our knowledge, this is the first description of an array-based assessment of peroxidase activity of G-quadruplex-hemin complexes. By using this novel assay system, more than 200 different mutants were found that had significantly higher activities than the original DNAzyme sequence. This microarray-based DNAzyme evaluation system is useful for identifying highly active new DNAzymes that might have potential as tools for developing DNA-based biosensors with aptamers.

Polymeric membrane neutral phenol-sensitive electrodes for potentiometric G-quadruplex/hemin DNAzyme-based biosensing.

The first potentiometric transducer for G-quadruplex/hemin DNAzyme-based biosensing has been developed by using potential responses of electrically neutral oligomeric phenols on polymeric membrane electrodes. In the presence of G-quadruplex/hemin DNAzyme and H(2)O(2), monomeric phenols (e.g., phenol, methylphenols, and methoxyphenols) can be condensed into oligomeric phenols. Because both substrates and products are nonionic under optimal pH conditions, these reactions are traditionally not considered in designing potentiometric biosensing schemes. However, in this paper, the electrically neutral oligomeric phenols have been found to induce highly sensitive potential responses on quaternary ammonium salt-doped polymeric membrane electrodes owing to their high lipophilicities. In contrast, the potential responses to monomeric phenolic substrates are rather low. Thus, the G-quadruplex/hemin DNAzyme-catalyzed oxidative coupling of monomeric phenols can induce large potential signals, and the catalytic activities of DNAzymes can be probed. A comparison of potential responses induced by peroxidations of 13 monomeric phenols indicates that p-methoxyphenol is the most efficient substrate for potentiometric detection of G-quadruplex/hemin DNAzymes. Finally, two label-free and separation-free potentiometric DNA assay protocols based on the G-quadruplex/hemin DNAzyme have been developed with sensitivities higher than those of colorimetric and fluorometric methods. Coupled with other features such as reliable instrumentation, low cost, ease of miniaturization, and resistance to color and turbid interferences, the proposed polymeric membrane-based potentiometric sensor promises to be a competitive transducer for peroxidase-mimicking DNAzyme-involved biosensing.

A novel dot-blot DNAzyme-linked aptamer assay for protein detection.

In this work, a novel dot-blot DNAzyme-linked aptamer assay (DLAA) for protein detection is developed with thrombin as a model protein. A peroxidase-like DNAzyme which serves as the catalytic label is tethered to a 15-mer thrombin-binding aptamer to form a label-free DNAzyme-linked aptamer probe. Based on specific interaction of the aptamer with target protein immobilized on nitrocellulose membrane, a DNAzyme layer is introduced onto the membrane. The DNAzyme can catalyze the H(2)O(2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine to produce a colored insoluble product that is apt to be adsorbed onto the nitrocellulose membrane. As a result, blue dots appear on the membrane, in contrast to the colorless background. As the concentration of thrombin increases, the color of dots gets deep. Such a protein concentration-dependent color change can be quantified via an image-processing software, with a detection limit of 0.6 microM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis.

Analysis of DNA and single-base mutations using magnetic particles for purification, amplification and DNAzyme detection.

The amplified detection of DNA or of single-base mismatches in DNA is achieved by the use of nucleic acid-functionalized magnetic particles that separate the recognition duplexes and, upon amplification, yield chemiluminescence-generating DNAzymes as reporter units. The analysis of M13 phage ssDNA is achieved by the hybridization of the analyte to capture nucleic acid-functionalized magnetic particles followed by the binding of a DNA machine unit to the analyte domain. The magnetic separation of the multi-component-functionalized magnetic particles, followed by their reaction with polymerase, dNTPs, and the nicking enzyme (Nb.BbvCI) activate the autonomous synthesis of the horseradish peroxidase-mimicking DNAzyme that acts as chemiluminescent reporter. The single-base mutation in DNA is achieved by coupling of the DNA machine to the mutant DNA/capture nucleic acid-functionalized magnetic particles hybrid structure. The activation of the polymerization/nicking cycles yield the chemiluminescent reporting DNAzyme. The magnetic separation of the DNA recognition hybrids improves the signal-to-noise ratio of the analytical protocol as compared to related DNAzyme synthesizing schemes.

Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers.

A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The design was aided by the FRET study that showed the dissociated substrate maintained a random coil conformation with an end-to-end distance of approximately 39 A, which is much shorter than that of the fully extended DNA. With this new design, the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design. This suppression of background fluorescence signals was achieved without losing selectivity of the sensor. The new design makes it possible to use the sensor for practical applications in a wide temperature range. The design principle presented here should be applicable to other nucleic acid-based biosensors to decrease background fluorescence.

Identification of cleavage sites in the HIV-1 TAR RNA by 10-23 and 8-17 catalytic motif containing DNA enzymes.

A quick identification of a cleavage site in the target RNA molecule to obtain sequence-specific cleavage by either catalytic RNA (ribozymes) or DNA (DNA enzymes) is very important for achieving gene-specific suppression. These molecules could also provide important information on the secondary and tertiary structure of the target RNA molecule. We have exploited the use of two kinds of DNA enzymes, namely, the 10-23 and 8-17 catalytic motif containing DNA enzymes, to achieve these objectives. We identified several DNA enzyme cleavage sites in the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) RNA-a structural feature present at the 5' end of all HIV-1 transcripts. Most of the DNA enzymes that cleaved the TAR RNA were targeted to the regions that were single-stranded in the predicted structure. Regions that were predicted to be base-paired (stem) failed to show any detectable cleavage. The DNA enzyme possessing the 8-17 catalytic motif was extremely efficient in cleaving full length, as well as short, HIV-1 specific transcripts. The efficiency of cleavage of the same target RNA by DNA enzymes that possessed the 10-23 catalytic motif was significantly less in comparison, and they failed to cleave the short transcripts. These molecules, in principle, have the potential to down regulate expression of all HIV-1 transcripts from a wide range of isolates because this region is functionally very well conserved.