Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.     

Real time monitoring uracil excision using uracil-containing molecular beacons

As a highly conserved damage repair protein, UDG excises uracil bases through its glycosylase activity. We report here an alternative fluorescence method for UDG assay with high accuracy and sensitivity by applying uracil-modified molecular beacons as substrates. The detection limit of UDG is 0.005 U mL⁻¹. The K_M and k_cat are 0.89 ± 0.1 μM and 210 ± 10 min⁻¹, respectively. The method is applied to screening inhibitors and the results indicate that both of the 5-FU and cisplatin can inhibit UDG activity with the IC₅₀ values of 6.1 ± 0.52 mM and 3.2 ± 0.24 mM, respectively. Furthermore, the combination of uracil-modified molecular beacons and nuclease inhibitor makes the new method possible to specifically detect UDG activity in cell-free extracts and serum. Taken together, the simple, rapid and sensitive method has potential relevance for a variety of applications, such as molecular diagnosis and screening of UDG inhibitors.     

Real-time monitoring of DNAzyme cleavage process using fluorescent assay

Detection of deoxyribozyme(DNAzyme) cleavage process usually needs complex and time-consuming radial labeling,gel electrophoresis and autoradiography.This paper reported an approach to detect DNAzyme cleavage process in real time using a fluorescence probe.The probe was employed as DNAzyme substrate to convert directly the cleavage information into fluorescence signal in real time.Compared with traditional approach,this non-isotope method not only brought a convenient means to monitor the DNAzyme cleavag...     

Chimeric peptide beacons: a direct polypeptide analog of DNA molecular beacons

We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.     

Quadruplex-based molecular beacons as tunable DNA probes

Molecular beacons (MBs) are fluorescent nucleic acid probes with a hairpin-shaped structure in which the 5' and 3' ends are self-complementary. Due to a change in their emissive properties upon recognition with complementary sequences, MBs allow the diagnosis of single-stranded DNA or RNA with high mismatch discrimination, in vitro and in vivo. Whereas the stems of MB hairpins usually rely on the formation of a Watson-Crick duplex, we demonstrate in this report that the preceding structure can be replaced by a G-quadruplex motif (G4). Intramolecular quadruplexes may still be formed with a central loop composed of 12 to 21 bases, therefore extending the sequence repertoire of quadruplex formation. G4-MB can efficiently be used for oligonucleotide discrimination: in the presence of a complementary sequence, the central loop hybridizes and forms a duplex that causes opening of the quadruplex stem. The corresponding G4-MB unfolding can be detected by a change in its fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using G4-MB instead of traditional MB. In particular, the intrinsic feature of the quadruplex motif facilitates the design of functional molecular beacons by independently varying the concentration of monovalent or divalent cations in the medium.     

Real-time surface-enhanced Raman spectroscopy monitoring of surface pH during electrochemical melting of double-stranded DNA

The application of a negative potential ramp at a double-stranded DNA (dsDNA) functionalized electrode surface results in the gradual denaturation of the DNA in a process known as electrochemical melting. The underlying physical chemistry behind electrochemically driven DNA denaturation is not well understood, and one possible mechanism is a change in local pH at the electrode surface. We demonstrate that by coimmobilization of p-mercaptobenozic acid at a dsDNA-functionalized electrode surface, it is possible to monitor both DNA denaturation and the local pH simultaneously using surface-enhanced Raman spectroscopy. We find that the local pH at the electrode surface does not change as the applied potential is scanned negative and the dsDNA denatures. We therefore conclude that in these experiments electrochemical melting is not caused by electrochemically driven local pH changes.     

Real-time monitoring of enzymatic cleavage of nucleic acids using a quartz crystal microbalance.

The use of quartz crystal microbalance (QCM) for monitoring in situ the enzymatic cleavage of surface-confined nucleic acids by nucleases is described. Such real-time monitoring of mass changes associated with the enzymatic digestion indicates that the activity and specificity of nucleases is preserved at the gold surface, and can be used for manipulating surface-confined DNAs and RNAs. These observations indicate great promise for using QCM for elucidating the interactions of nucleic acids with enzymes, and for enhancing the power of hybridization biosensors.     

Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

Locked nucleic acid molecular beacons

A novel LNA-MB (molecular beacon based on locked nucleic acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.     

Polyvalent carbocyanine molecular beacons for molecular recognitions

Polyvalent carboxylate-terminating near-infrared (NIR) carbocyanine molecular beacons were prepared by homologation of reactive carboxyl groups of the beacon with imino diacetic acid. Their conjugation with unprotected d-(+)-glucosamine gave dendritic arrays of the carbohydrate on an inner NIR chromophore core. In vivo evaluation of the dendritic glucosamine constructs shows enhanced uptake in proliferating tumor cells relative to surrounding normal tissue. The structural framework of these polyvalent beacons permits the amplification by synergistic effects of a variety of bioactive motifs or chemical sensors in molecular recognition interactions without dramatic change of their desirable NIR spectral properties.     

Biosensors based on molecular beacons

A fundamental molecular beacon (MB) consists of a special short nucleic acid strand with a fluorophore-quencher pair attached to its ends. It provides a unique framework that is susceptible to conformational transitions between a hairpin (closed) conformation and an extended (open) conformation. These two conformations are readily discernible because of their differing fluorescence emission characteristics. The broad applicability of the robust MB sensing platform has attracted widespread interest, resulting in extensive research studies ranging from theoretical and bioanalytical chemistry to molecular biology and biomedical applications. In this paper, the principles of MB design and the modes and mechanisms of MB operation are reviewed, including MB modifications based on the utilisation of a thymidine-thymidine mismatch in hybridised MB stem, aptamers, peptides and locked nucleic acid strands in an MB loop, as well as plasmonic quenchers, quantum dots and interactions with graphene and graphene oxide. Specific applications of MBs in the analysis of enzymes, DNA mutation, phosphorylation, methylation and ligation, followed by the detection of pathogens and applications in cancer and other disease diagnostics and therapeutics are also reviewed. Molecular beacon-based sensing platforms are expanding rapidly and offer a promising bioanalytical tool for inexpensive and reliable analysis for research and field diagnostics.     

Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel

A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described. A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy (SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising real-time detection method for label-free DNA targets in the solution phase. Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel     

Real-time monitoring of strand-displacement DNA amplification by a contactless electrochemical microsystem using interdigitated electrodes

This paper reports the design and implementation of a contactless conductivity detection system which combines a thermal control cell, a data processing system and an electrochemical (EC) cell for label-free isothermal nucleic acid amplification and real-time monitoring. The EC cell consists of a microchamber and interdigitated electrodes as the contactless conductivity biosensor with a cover slip as insulation. In our work, contactless EC measurements, the effects of trehalose on amplification, and chip surface treatment are investigated. With the superior performance of the biosensor, the device can detect the amount of pure DNA at concentrations less than 0.1 pg μl(-1). The EC cell, integrated with a heater and a temperature sensor, has successfully implemented nicking-based strand-displacement amplification at an initial concentration of 2.5 μM and the yields are monitored directly (dismissing the use of probes or labels) on-line. This contactless detector carries important advantages: high anti-interference capability, long detector life, high reusability and low cost. In addition, the small size, low power consumption and portability of the detection cell give the system the potential to be highly integrated for use in field service and point of care applications.     

Label-free monitoring of site-specific DNA cleavage by EcoRI endonuclease using cyclic voltammetry and electrochemical impedance

Cyclic voltammetry (CV) combined with electrochemical impedance spectroscopy (EIS) were proposed to monitor the site-specific DNA cleavage by EcoRI endonuclease without using external label. The alteration of CV and EIS signal demonstrated that double-strands (dsDNA) contain recognition sequence was cleaved by EcoRI endonuclease. Real-time monitoring indicated that the dsDNA was cleaved by EcoRI more than 90% after 2 h of enzyme digestion time. Control experiment showed that the DNA cleavage by EcoRI endonuclease is site-specific for DNA sequence. Experimental results demonstrated that the efficiency of EcoRI cleavage was highly dependent on the concentration of EcoRI concentration in the range from 0.04 to 0.4 U μL⁻¹ with one almost linear relationship.     

Real-time reaction monitoring using ion mobility-mass spectrometry

The potential of ion mobility (IM) spectrometry in combination with mass spectrometry (MS) for real-time reaction monitoring is reported. The combined IM-MS approach using electrospray ionization affords gas-phase analyte characterization based on both mass-to-charge (m/z) ratio and gas-phase ion mobility (drift time). The use of IM-MS analysis is demonstrated for the monitoring of the reaction products formed when 7-fluoro-6-hydroxy-2-methylindole is deprotonated by aqueous sodium hydroxide. Real-time reaction monitoring was carried out over a period of several hours, with the reaction mixture sampled and analysed at intervals of several minutes. Product ion relative intensity is enhanced selectively in the ion mobility-selected mass spectrum, compared to mass spectrometry alone. The combined IM-MS approach has potential as a rapid and selective technique to aid pharmaceutical process control and for the elucidation of reaction mechanism.     

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.     

Real-time characterization of cytotoxicity using single-cell impedance monitoring

Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.     

Real-time monitoring of polyurethane production using near-infrared spectroscopy

A process near-infrared (NIR) spectrophotometer was interfaced directly to a reactor by using a fiber optic bundle interactance immersion probe. This remote sensor configuration enables the production of polyurethanes to be monitored in real-time. A Beer's Law model was derived for the quantitative determination of isocyanate in the urethane polymerization reaction. Statistical process control was used to observe trends in the polymerization reaction. The integration of NIR process analytical instrumentation directly into the process provides real-time chemical information that yields improvements in product quality and consistency, while minimizing reaction time.