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.     

In vitro selection of molecular beacons

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.     

Imaging native beta-actin mRNA in motile fibroblasts

Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label beta-actin mRNA in living and motile chicken embryonic fibroblasts. beta-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of beta-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing beta-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of beta-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.     

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     

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.     

Study of the mechanism of TCR antagonism using dual-TCR-expressing T cells

The mechanism of action of TCR antagonists is incompletely understood. T cells expressing two distinct TCRs have been used to test competition for TCR occupancy as a potential mechanism. Previous studies with CD4 T cells showed that an antagonist for one TCR inhibited the response to the other TCR (cross-antagonism), whereas studies with CD8 cells failed to demonstrate cross-antagonism. To determine whether CD4 and CD8 cells were intrinsically different or whether the differences were the result of the use of different effector assays, we studied both CD4 and CD8 dual-TCR-expressing T cells. In the CD4 system, consistent with previous reports, cross-antagonism of proliferation was observed. In the CD8 system, cross-antagonism was observed using proliferation as readout but not when target cell cytolysis was used. These results suggest that different mechanisms may be involved in the inhibition of proliferation and inhibition of cytotoxic effector function, the latter only involving competition for TCR occupancy. Inhibition of proliferation appears to be more complex and other mechanisms such as sequestration of signaling molecules or negative signaling may be involved. The fact that 10- to 20-fold more antagonist was needed to achieve cross-antagonism compared with inhibition of the cognate TCR is consistent with the hypothesis that competition for TCR occupancy is also a major, albeit not sole, mechanism of antagonism of the proliferative responses of CD4 and CD8 cells.     

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.     

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.     

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.     

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 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.     

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).     

Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons

Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 x 10(3.6) 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.     

The importance of including imperfect detection models in eDNA experimental design

Environmental DNA (eDNA) is DNA that has been isolated from field samples, and it is increasingly used to infer the presence or absence of particular species in an ecosystem. However, the combination of sampling procedures and subsequent molecular amplification of eDNA can lead to spurious results. As such, it is imperative that eDNA studies include a statistical framework for interpreting eDNA presence/absence data. We reviewed published literature for studies that utilized eDNA where the species density was known and compared the probability of detecting the focal species to the sampling and analysis protocols. Although biomass of the target species and the volume per sample did not impact detectability, the number of field replicates and number of samples from each replicate were positively related to detection. Additionally, increased number of PCR replicates and increased primer specificity significantly increased detectability. Accordingly, we advocate for increased use of occupancy modelling as a method to incorporate effects of sampling effort and PCR sensitivity in eDNA study design. Based on simulation results and the hierarchical nature of occupancy models, we suggest that field replicates, as opposed to molecular replicates, result in better detection probabilities of target species.     

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.     

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.     

Multiplex detection of single-nucleotide variations using molecular beacons

We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis.     

Transport and detection of unlabeled nucleotide targets by microtubules functionalized with molecular beacons

Shrinking biosensors down to microscale dimensions enables increases in sensitivity and the ability to analyze minute samples such as the contents of individual cells. The goal of the present study is to create mobile microscale biosensors by attaching molecular beacons to microtubules and using kinesin molecular motors to transport these functionalized microtubules across two-dimensional surfaces. Previous work has shown that microfluidic channels can be functionalized with kinesin motors such that microtubules can be transported and directed through these channels without the need for external power or pressure-driven pumping. In this work, we show that molecular beacons can be attached to microtubules such that both the fluorescence reporting capability of the beacon and the motility of the microtubules are retained. These molecular beacon-functionalized microtubules were able to bind ssDNA target sequences, transport them across surfaces, and report their presence by an increase in fluorescence that was detected by fluorescence microscopy. This work is an important step toward creating hybrid microdevices for sensitive virus detection or analyzing mRNA profiles of individual cells.     

Shedding light on health and disease using molecular beacons.

The detection and identification of pathogens is often painstaking due to the low abundance of diseased cells in clinical samples. The genomic sequences of the pathogen can be amplified through methods such as the polymerase chain reaction and nucleic acid sequence-based amplification, but the nucleic acid targets are often lost among other unintended products of amplification. Novel nucleic acid probes known as molecular beacons have been developed allowing for the rapid and specific detection of genetic markers of a disease. Molecular beacons are hairpin-forming oligonucleotides labelled at one end with a quencher and at the other end with a fluorescent reporter dye. In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridisation, resulting in the restoration of fluorescence. The ability to transduce target recognition into a fluorescence signal with high signal-to-background ratio, coupled with an improved specificity, has allowed molecular beacons to enjoy a wide range of biological and biomedical applications. Here, we describe the basic features of molecular beacons, review their applications in disease detection and diagnosis and discuss some of the issues and challenges of in vivo studies. The aim of this paper is to foster the development of new molecular beacon-based assays and to stimulate the application of this technology in laboratory and clinical studies of health and disease.