Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.     

Hybridization kinetics and thermodynamics of molecular beacons

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem–loop structure of molecular beacons provides a competing reaction for probe–target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure–function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon–target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.     

Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

PNA beacons for duplex DNA

We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.     

Structure-function relationships of shared-stem and conventional molecular beacons

Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.     

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.     

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7 that eliminates the risk of a false negative signal

A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the F?rster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.     

Single-mismatch detection using gold-quenched fluorescent oligonucleotides

Here we describe a hybrid material composed of a single-stranded DNA (ssDNA) molecule, a 1.4 nm diameter gold nanoparticle, and a fluorophore that is highly quenched by the nanoparticle through a distance-dependent process. The fluorescence of this hybrid molecule increases by a factor of as much as several thousand as it binds to a complementary ssDNA. We show that this composite molecule is a different type of molecular beacon with a sensitivity enhanced up to 100-fold. In competitive hybridization assays, the ability to detect single mismatch is eightfold greater with this probe than with other molecular beacons.     

Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions

Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to submicrometer sizes, have been developed for DNA/RNA analysis. The fluorescence-based biosensors have been applied in DNA/ RNA detection without the need for a dye-labeled target molecule or an intercalation reagent in the testing solution. Molecular beacons are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. We have designed a surface-immobilizable biotinylated ssDNA molecular beacon for DNA hybridization at a liquid-solid interface. The MBs have been immobilized onto ultrasmall optical fiber probes through avidin-biotin binding. The MB DNA biosensor has been used directly to detect, in real time, its target DNA molecules without the need for a competitive assay. The biosensor is stable and reproducible. The MB DNA biosensor has selectivity with single base-pair mismatch identification capability. The concentration detection limits and mass detection limits are 0.3 nM and 15 amol for a 105-microm biosensor, and 10 nM and 0.27 amol for a submicrometer biosensor, respectively. We have also prepared molecular beacon DNA biosensor arrays for simultaneous analysis of multiple DNA sequences in the same solution. The newly developed DNA biosensors have been used for the precise quantification of a specific rat gamma-actin mRNA sequence amplified by the polymerase chain reaction.     

Detection of Transgenes in Crop Plants Using Molecular Beacon Assays

Molecular beacons are oligonucleotide probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary targets, they undergo a conformational transition that turns on their fluorescence. These probes recognise their targets with higher specificity than linear probes and can easily discriminate targets that differ from one another by a single nucleotide. As a model system to test the applicability of molecular beacons in crop plants, we have designed a molecular beacon to detect the bar transgene in barley. Results from this experiment indicate that molecular beacons can be successfully employed in detecting transgenes, simultaneously combining the benefits of being highly reproducible and sensitive. The molecular beacon assay is suitable for diagnostics, simultaneously being employed in the development of rapid DNA-based assays for analysing single nucleotide polymorphisms (SNPs).     

Molecular beacon sequence design algorithm

A method based on Web-based tools is presented to design optimally functioning molecular beacons. Molecular beacons, fluorogenic hybridization probes, are a powerful tool for the rapid and specific detection of a particular nucleic acid sequence. However, their synthesis costs can be considerable. Since molecular beacon performance is based on its sequence, it is imperative to rationally design an optimal sequence before synthesis. The algorithm presented here uses simple Microsoft Excel formulas and macros to rank candidate sequences. This analysis is carried out using mfold structural predictions along with other free Web-based tools. For smaller laboratories where molecular beacons are not the focus of research, the public domain algorithm described here may be usefully employed to aid in molecular beacon design.     

Microbial Detection with Low Molecular Weight RNA

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%. Received: 13 March 2001 / Accepted: 3 May 2001     

Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA

Traditional methods to assay enzymatic cleavage of DNA are discontinuous and time consuming. In contrast, recently developed fluorescence methods are continuous and convenient. However, no fluorescence method has been developed for single-stranded DNA digestion. Here we introduce a novel method, based on molecular beacons, to assay single-stranded DNA cleavage by single strand-specific nucleases. A molecular beacon, a hairpin-shaped DNA probe labeled with a fluorophore and a quencher, is used as the substrate and enzymatic cleavage leads to fluorescence enhancement in the molecular beacon. This method permits real time detection of DNA cleavage and makes it easy to characterize the activity of DNA nucleases and to study the steady-state cleavage reaction kinetics. The excellent sensitivity, reproducibility and convenience will enable molecular beacons to be widely useful for the study of single-stranded DNA cleaving reactions.     

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.     

Target discrimination by surface-immobilized molecular beacons designed to detect Francisella tularensis

A molecular beacon (MB) array was designed based on unique regions of the 16S rRNA of the bacterium Francisella tularensis. Nucleic acid molecular beacons undergo a spontaneous fluorogenic conformational change when they hybridize to specific complementary targets. The array was printed on aldehyde glass or hydrogel slides and evaluated for functioning in presence of complementary oligonucleotide sequences, single-nucleotide mismatch sequences and multiple nucleotide mismatch sequences. Discriminating true target from mismatched targets was found to be dependent on type, number, and location of mismatches within the beacon (i.e. located in the stem or loop regions). Optimal conditions for molecular beacon deposition, and target hybridization were determined for oligonucleotide target mismatch discrimination. The beacon array was stable upon recharging by exposure to an alkaline solution, and repeatedly used. In addition, performance of the beacon array biosensor was compared with molecular beacons in homogeneous solution.     

A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics.

We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets. The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Reaction of AE with sodium sulfite renders it non-chemiluminescent. When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent. In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent. Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence. Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets. The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques.     

Tiny Molecular Beacons: LNA/2'-O-methyl RNA Chimeric Probes for Imaging Dynamic mRNA Processes in Living Cells

New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2'-O-methyl RNA chimera oligonucleotides "tiny molecular beacons". We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons and the flexibility to couple them to a large variety of fluorophores and quenchers render them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for oskar mRNA and microinjected them into living Drosophila melanogaster oocytes. We then imaged the live oocytes via spinning disk confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons and are able to access previously inaccessible target regions.     

Biomolecular computation with molecular beacons for quantitative analysis of target nucleic acids

Molecular beacons are efficient and useful tools for quantitative detection of specific target nucleic acids. Thanks to their simple protocol, molecular beacons have great potential as substrates for biomolecular computing. Here we present a molecular beacon-based biomolecular computing method for quantitative detection and analysis of target nucleic acids. Whereas the conventional quantitative assays using fluorescent dyes have been designed for single target detection or multiplexed detection, the proposed method enables us not only to detect multiple targets but also to compute their quantitative information by weighted-sum of the targets. The detection and computation are performed on a molecular level simultaneously, and the outputs are detected as fluorescence signals. Experimental results show the feasibility and effectiveness of our weighted detection and linear combination method using molecular beacons. Our method can serve as a primitive operation of molecular pattern analysis, and we demonstrate successful binary classifications of molecular patterns made of synthetic oligonucleotide DNA molecules.     

Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film

We developed a new technique to immobilize a set of molecular beacons on an agarose film-coated slide and found that it has the ability to identify a single nucleotide difference in label-free DNA targets. The annealing properties, specificity and hybridization dynamics of the present technique were compared with those of the conventional technique that directly immobilizes molecular beacons on a planar glass slide. It is demonstrated that the molecular beacon array on an agarose film has high quench efficiency, an excellent discrimination ratio for single nucleotide mismatches and a short detection time. We hypothesize that such a low fluorescence background and high specificity molecular beacon array will find practical applications in label-free, high-throughput mutation analysis and disease diagnosis.