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.     

Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons

Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.     

Identification of high-stringency DNA hairpin probes by partial gene folding

Hairpin DNA sequences are widely used as probes for oligonucleotides in a broad range of assays, often as "molecular beacons". A potential disadvantage of the standard methodology for molecular beacon design is the need to add several self-complementary bases to each end of the probe, since these do not correspond to the target sequence. We describe a conceptually new method of hairpin DNA probe identification, in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within an expressed gene having the requisite hairpin structure. Intuitively, such probes should have significantly improved performance over "traditional" hairpin probes, because they are fully complementary with the target. We present experimental evidence verifying this hypothesis for a series of hairpin probes targeting the pag gene of Bacillus anthracis.     

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.     

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.     

Amplified detection of nucleic acid by G-quadruplex based hybridization chain reaction

A protein-free, isothermal, self-amplified nucleic acid sensing system which was a G-quadruplex integrated hybridization chain reaction (GQ-HCR) system was developed. The G-quadruplex was closed two-thirds in the loop and one-third in the stem of one of the GQ-HCR hairpin probes. In the absence of the target molecule, the GQ-HCR probes stayed as inactive meta-stable hairpin structures and the G-quadruplex was inert. Reversely, the GQ-HCR probes could be cross-opened to start a hybridization chain reaction and the closed G-quadruplex could be released to be free when the GQ-HCR probes came across the target molecule. The GQ-HCR nucleic acid sensing system could detect as low as 7.5nM ssDNA or RNA by the colorimetric method and 4nM ssDNA by the fluorometric method. Less than 10 copies of dsDNA template could also be detected when PCR was combined with the GQ-HCR system (PCR+GQ-HCR). Because of these advantages, the GQ-HCR system was also studied for application in visual chip detection to obtain a satisfactory repeatable and specific result.     

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.     

Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.     

Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology

The specific structural features of stem-loop (hairpin) DNA constructs provide increased specificity of target recognition. Recently, several robust assays have been developed that exploit the potential of structurally constrained oligonucleotides to hybridize with their cognate targets. Here, I review new diagnostic approaches based on the formation of stem-loop DNA oligonucleotides: molecular beacon methodology, suppression PCR approaches and the use of hairpin probes in DNA microarrays. The advantages of these techniques over existing ones for sequence-specific DNA detection, amplification and manipulation are discussed.     

Tripartite molecular beacons

Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications.     

Immobilized stem-loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA

We have designed and evaluated novel DNA stem-loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem-loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem-loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection.     

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.     

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.     

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.     

Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure

Molecular beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramolecular heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the respective positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Förster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its associated absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of molecular beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.     

Progress in designing nucleic acid structures and fine-tuning their interactions

The prediction of the structure of biological macromolecules at the atomic level and the design of new meta-stable structures and secondary interactions are critical tests of our understanding of the structures and the inter-atomic forces that underlie molecular biology. The capacity to accurately predict and design new structures and interactions will allow us to create nucleic acid sequences that will fold in new and useful ways. Here, we present some results to demonstrate the progress we have made in designing and assembling new nucleic acid structures that will make an increasingly important contribution to biology and medicine. We call the reaction cycle that exemplifies our approach 'A handshake from a hairpin on the way to a double helix.'     

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem–loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity.     

Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp

An electrochemically amplified molecular beacon (EAMB) biosensor is constructed using thiolated hairpin DNA-ferrocene probes on gold electrode. The switching from "on" to "off" states of individual probes in the presence of complementary DNA target influences the electrode potential, besides the current, owing to changes in surface density of the electroactive hairpin DNA-ferrocene probes. The EAMB biosensor demonstrates linear range over 8 orders of magnitude with ultrasensitive detection limit of 2.3 × 10(-14)M for the quantification of a 21-mer DNA sequence. Its applicability is tested against PCR amplicons derived from genomic DNA of live Legionella pneumophila. Excellent specificity down to one and three nucleotides mismatches in another strain of L. pneumophila and a different bacterium species, respectively, is demonstrated.     

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T₂-biotin·dT-T₂ loop. The third base was a biotinylated uracil (U_B) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3′ dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3′ end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5′-FITC, or radiolabeled with [γ-³³P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.