When You Wish Upon A Star: Molecular Beacons: Real Time in a Twinkle

Table of Licensed Providers of Molecular Beacons and Kits Using molecular beacons for spectral genotyping. Differently-colored molecular probes specific for the wild-type and mutant alleles are designed. DNA amplified from homozygous wild-type individuals binds only to the fluorescein-labeled molecular beacons (left). DNA from homozygous mutants binds only the tetramethylrhodamine-labeled molecular beacons (right). Both types of molecular probes will bind to amplicons generated from the DNA of heterozygous individuals (center). Reprinted with permission from L.G. Kostrikis et al., "Spectral genotyping of human alleles," Science 279,1228-9, 1998. Copyright 1998, American Association for the Advancement of Science The annealing of a nucleic acid strand to its complement is one of the most specific molecular recognition events known. A plethora of approaches exploiting this fact have been used for basic research. For example, nucleic acid blotting techniques have been used to make great strides in our understanding of gene organization and function. In these techniques, DNA fragments are immobilized on a solid support. A labeled oligonucleotide probe containing the sequence of interest is then used to detect its counterpart. In today's laboratories, nucleic acid techniques are increasingly being harnessed for use in practical applications, such as the molecular diagnosis of disease. But what if you need to monitor the synthesis of nucleic acids as it is happening? Or what if your research calls for the labeling of nucleic acids within living cells? Methods based on the immobilization of hybridized nucleic acids or other means to isolate probe-target complexes cannot be used for these applications. When one is detecting nucleic acids by hybridization, there are numerous situations in which it is not desirable to isolate probe-target complexes from the excess of probes that are used. Sanjay Tyagi and Fred Russell Kramer, scientists at the Public Health Research Institute, N. Y., developed a new class of probe to be used just for those types of applications. Termed "molecular beacons," these single-stranded oligonucleotide probes can be used to report the presence of specific nucleic acids in solution. These probes can monitor nucleic acid synthesis in real time, without interrupting the reactions, and can be used to locate specific RNAs within living cells without killing the cells. Molecular beacons are hairpin-shaped ("stem-and-loop") molecules with an internally quenched fluorophore. They are "dark" when free in solution. Fluorescence is restored upon binding to the target nucleic acid. The loop portion of a molecular beacon is a probe sequence complementary to a target nucleic acid molecule. The arm sequences flanking either side of the probe are complementary to each other but are unrelated to the target sequence. These arm sequences anneal to form the molecule's stem. A fluorescent moiety is attached to the end of one arm, and a quenching moiety is attached to the end of the other. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. In brief, the quenching moiety dissipates the energy that it receives from the fluorophore as heat, rather than emitting it as light. Thus, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem. The molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart. The fluorophore and quencher are moved away from each other, leading to the restoration of fluorescence. Unhybridized molecular beacons do not fluoresce; thus, it is not necessary to remove them to observe hybridized probes. The usefulness of "detection without separation" for certain applications, such as real-time monitoring of polymerase chain reactions (PCR), cannot be overemphasized. This feature enables the synthesis of nucleic acids to be monitored as it is occurring, in sealed tubes, without additional manipulations. In PCR applications, the probe portion of the molecular beacons is complementary to a sequence in the middle of the expected amplicon. The length of the loop sequence (10-40 nucleotides) should be chosen so that the probe-target hybrid is stable at the annealing temperature. The length of the arm sequences (5-7 nucleotides) should be chosen so that a stem is formed at the annealing temperature of the polymerase chain reaction. Also, the stem sequence must be designed to ensure that the two arms hybridize to each other but not to the probe sequences. Molecular beacons with appropriate thermal denaturation characteristics are included in each reaction at a concentration similar to that of the primers. During the denaturation step, molecular beacons assume a random coil configuration and fluoresce. Their stems "open and shine" when the temperature is higher than their melting point. However, it's "lights out" when the temperature drops below the melting point. As the temperature is lowered to allow primer annealing, the molecular beacons renature with lightning speed. The reformation of intramolecular hairpins takes less than a microsecond, which is a substantially shorter time than it takes to change the temperature of the solution. When the temperature becomes favorable for hybridization, the molecular beacons will also interact with target strands, generating fluorescence. As the target strands synthesized in a reaction accumulate, the fraction of molecular beacons that are bound to targets increases, causing a brighter fluorescent signal. When the temperature is raised to allow primer extension, the molecular beacons dissociate from their targets. Thus, the molecular beacons do not interfere with the DNA polymerization reaction. A new hybridization takes place in the annealing step of every cycle. During the linear phase of the reaction, the intensity of the resulting fluorescence correlates with the amount of accumulated amplicon. Tyagi, Kramer, and colleagues have characterized molecular beacons containing a number of different fluorophore-quencher combinations. An ideal fluorophore-quencher pair would be completely unable to fluoresce when the two components are in close proximity. In their early studies, these scientists found that the fluorophore 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) and the quencher 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) were a great team. When stimulated by ultraviolet light with a peak wavelength of 336 nm, EDANS emits a brilliant blue fluorescence with a peak wavelength of 490 nm. DABCYL is a nonfluorescent chromophore with an absorption spectrum closely overlapping the emission spectrum of EDANS. When acting solo, EDANS first captures and stores radiant energy of the appropriate wavelength. Then, in just a few nanoseconds, EDANS emits that energy in the form of light of a longer wavelength. A different scenario occurs when EDANS is in close proximity to an appropriate quencher, such as DABCYL. When EDANS and DABCYL are sufficiently close to one another, the energy stored in EDANS is transferred to DABCYL and dissipated as heat. Because the energy is released as heat, rather than light, the fluorescence is said to be quenched. As a pair, EDANS and DABCYL exhibit properties that are particularly advantageous for their inclusion in molecular beacons. The distance within which DABCYL is able to quench the fluorescence of EDANS is small compared to the distance that separates them after hybridization to the target has occurred. This greatly diminishes the likelihood of problems with background signal. Furthermore, EDANS is negatively charged and hydrophilic, whereas DABCYL is neutral and hydrophobic. They neither strongly attract nor repel each other and therefore are not expected to alter the intrinsic stability of the stem hybrid. From a practical standpoint, an additional advantage is that a sulfhydryl-reactive form of EDANS and an amino-reactive form of DABCYL are commercially available. These moieties can be covalently linked to synthetic oligonucleotides via relatively simple procedures. Initially, Kramer's group chose fluorophore-quencher pairs with overlapping absorption/emission spectra. Such overlapping spectra are generally necessary for the transfer of the energy of a stimulated fluor to an acceptor molecule without the emission of light via a process termed fluorescence resonance energy transfer (FRET). Much to their surprise, these scientists found that DABCYL serves as a universal quencher for any fluorophore in molecular beacons. Quenching efficiency was independent of spectral overlap, and the efficiencies were higher than are usual for FRET. In molecular beacons, the fluorophore and the quencher are held so close together that direct transfer of energy is possible. The hairpin conformation holds the pair together in a nonfluorescent complex. Exploiting this feature, the scientists designed a set of molecular beacons employing DABCYL and different fluorophores. The emission spectra of this set spans the entire visible range, from blue to red. Molecular beacons are extraordinarily target-specific, ignoring nucleic acid target sequences that differ by as a little as a single nucleotide. This, along with the availability of different fluorophore-quencher pairs makes these probes extremely useful for multiplex applications. The detection of many different targets in the same solution can be achieved by the simultaneous use of several molecular beacons, each of which emits light of a different wavelength or which can be excited by light of a different wavelength. These characteristics also make molecular beacons exquisitely suited for use in the identification of, for example, genetic alleles (see Figure) or particular strains of infectious agents. Several companies offer molecular beacons and reagents for making them (see Table). Three basic types of products are available. Some vendors will prepare and purify the molecular beacon, complete with fluorophore and quencher. Some researchers prefer to purchase their oligonucleotide already conjugated to DABCYL but not the fluorophore. In this case, the oligonucleotide is premodified by DABCYL at the 3' end. The other arm will generally be modified to have a 5' amino- or thiol-reactive group, which enables the customer to couple it to his or her probe of choice. For even more flexibility, you can request that both ends of your oligonucleotide be modified with different 3'- and 5'- reactive groups. This enables total choice in the fluorophore and quencher used. For the complete do-it-yourselfer, supplies and reagents that enable you to modify the ends of oligonucleotides in your own laboratory are also commercially available for example from Glen Research (Sterling, Va.). A few recent references for molecular beacons are listed below. This, along with the company information summarized in the table, should help illuminate your way--whether you are considering using molecular beacons in the near future, or just curious about this "hot" new technique. S. Tyagi and F.R. Kramer, "Molecular beacons: probes that fluoresce upon hybridization," Nature Biotechnology, 14: 303-8, 1996. S. Tyagi et al., "Multicolor molecular beacons for allele discrimination," Nature Biotechnology, 16: 49-53, 1998. A.S. Paitek et al., "Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis," Nature Biotechnology 16: 359-63, 1998. L.G. Kostrikis et al., "Spectral genotyping of human alleles," Science 279: 1228-9, 1998. The author, Deborah Wilkinson, can be reached at deborahwilkinson@sprynet.com Table of Licensed Providers of Molecular Beacons and Kits

When You Wish Upon A Star: Molecular Beacons: Real Time in a Twinkle

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