Probing the darkness

ILLUSTRATION: TONY FERNANDEZ

The wealth of genetic information generated in the past decade has enabled researchers and clinicians to answer questions on topics ranging from gene identity, expression, and mutation to molecular diagnostics and drug development. A variety of genetic techniques has been used, such as the polymerase chain reaction (PCR), electrophoretic gel blotting, and sequencing, but each technique has its own challenges in the areas of quantitation, specificity, and through-put. Although the standard methods have been adequate for developing qualitative answers, the time is ripe for quantitative answers; and the old tools poorly address the new questions.

As Robert Georgantis said in “Real-time results” (Modern Drug Discovery, July 2001, p 57), “Quantitative PCR and [reverse transcription] PCR rely on the concept that the amount of PCR product present after each amplification cycle is directly related to the number of target copies originally present in the sample.”

Traditionally, scientists measured the amount of PCR product by treating the samples with intercalating fluorescent dyes such as ethidium bromide or SYBR Green, but these dyes lacked specificity and merely told the researcher how much DNA resided in the sample. To get around this problem, researchers introduced oligonucleotides bearing fluorescent tags (fluorophores) and containing sequences complementary to that of the target DNA (e.g., TaqMan probes). But impure samples suffered from problems of degradation, and unbound fluorescent markers caused high background.

Illuminating developments
In 1996, Sanjay Tyagi and colleagues at New York’s Public Health Research Institute took the concept of fluorescently labeled oligonucleotides one step further, introducing molecular beacons (MBs) (1). Like their predecessors, MBs carry a fluorophore (such as fluorescein or rhodamine) on one end of the oligonucleotide, but they also carry a fluorescence-quenching compound (typically DABCYL or EDANS) on the other (Figure 1A). Between the two compounds, the MB carries two complementary regions, 5–10 nucleotides long, that form a stem structure; the sequence complementary to the target DNA lies in a loop bounded by the stem.

When the MB is away from its target sequence, the stem structure holds the fluorophore and quenching groups close together, so that the quencher captures the light emitted by the fluorophore. Upon interacting with the target DNA, however, the stem structure is pulled apart, and the light from the fluorophore is detected spectrophotometrically. Thus, during a PCR reaction, MBs bind to the synthesized DNA such that the resulting level of fluorescence is directly correlated with the amount of target DNA. For researchers who do not wish to manufacture their own MBs, there are companies offering a wide array of fluorophores and quenchers (Table 1).

A few years later, Tom Brown and his colleagues at the University of Southampton (U.K.) added to the mix by introducing a new MB, which they dubbed the Scorpion primer because of the way it anneals to itself (2). The priming sequence that initiates the PCR is attached to the MB in such a way that it becomes part of the final PCR product (Figure 1B). The loop region of the MB then interacts with the newly synthesized strand of DNA, allowing the fluorescence to be detected. The Scorpion primer has an advantage over the typical MB in that its interaction with the target sequence is unimolecular, which improves the kinetics of the reaction.

Also seeking to improve MB technology, Weihong Tan and his colleagues at the University of Florida (Gainesville) and TriLink Biotechnologies (San Diego) went in another direction. They were concerned that MBs do not work in a precise “on-off” manner—the quenching of the fluorophore is never 100%—because of problems with emission-absorption profiles, the manner in which the MBs are synthesized, and the way in which the fluorophore and quencher are attached (3). To address these concerns, the researchers replaced the quencher with a second fluorophore (F₂). While in the stem–loop form, F₂ absorbs the light emitted by the first fluorophore (F₁) and emits its own light. But when the MB is bound to its target DNA, F₁ and F₂ are separated, and researchers can detect an increase in F₁ fluorescence and a decrease in F₂ fluorescence. The scientists argue that the ratio of the fluorescence intensity of F₁ to that of F₂ (I_F1/I_F2) provides a more accurate measure of the target DNA.

Beacon applications
As suggested earlier, one of the first techniques to use MBs was real-time PCR. Not only did MBs allow researchers to detect specific products (eliminating the detection of artifactual side products), but they also allowed a certain degree of discrimination with regard to point mutations that might occur during replication or were within the original sample. Such discrimination could be achieved because of the nature of the double-stranded stem and the fact that under the right conditions, intramolecular folding is energetically favored over the intermolecular association of an MB and a mutated target, which bind more weakly. In addition, by using different fluorophore–quencher combinations on MBs with different sequences, it is possible to detect up to four distinct DNA products. This process is known as multiplexing (4).

The ability of MBs to discriminate between sequences makes them an ideal tool for genetic screening and diagnostics. Researchers from The Netherlands recently used MBs and a robotic workstation to detect mutations in blood samples of a gene associated with cardiovascular disease, showing that the new technique worked as well as conventional methods and increased throughput (5). Similarly, Canadian researchers used MBs and the Cepheid (Sunnyvale, CA) Smart Cycler to develop an assay for distinguishing between two Candida species in blood samples (6). C. albicans and C. dubliniensis are virtually indistinguishable using microbiological methods and require entirely different patient treatment regimens.

Future applications
MBs are also moving, albeit slowly, into the chip world. Recently, Tan and Xiaojing Liu designed a biotinylated MB and bound it to the surface of a silica chip that had been chemically cross-linked with avidin, biotin’s binding partner (7). An excitation laser generated an evanescent field in a prism placed against the chip, and a charge-coupled device camera detected the resulting fluorescent signals. The researchers used this specialized biochip to detect concentrations reaching almost 1 nM, and they readily regenerated the chips for further use.

The development of MB biosensors also opens the door to the detection of compounds other than DNA, RNA, and peptide nucleic acid (PNA). Aptamers are oligonucleotides that have been selected to bind specifically to a variety of compounds ranging from proteins to small molecules; thus, Martin Stanton and his colleagues at Brandeis University (Waltham, MA) and the University of Texas at Austin combined MB and aptamer technologies to create highly specific protein detection devices (8).

The antithrombin aptamer forms a guanine quartet that acts as a scaffold to bring two connecting double-thymidine loops into direct contact with the thrombin (Figure 2A). The researchers speculated that by adding a complementary sequence to one end of the aptamer, they could disrupt the formation of the guanine scaffold. The scientists did this and, by adding a fluorophore and quencher on opposite ends of the aptamer, designed what they called an aptamer beacon (Figure 2B). In the absence of thrombin, the traditional MB stem–loop forms and the beacon is dark. But when thrombin is present, it stabilizes the formation of the guanine quartet, moving the fluorophore and quencher apart and thus creating a fluorescent signal. The protein–aptamer complex had an apparent K_d of ~10 nM.

The researchers recognize that the aptamer beacon system has some limitations, including the large number of proteins that bind to single-stranded DNA nonspecifically and interfere with the assays. They believe, however, that the technology has great potential because specific aptamers can be generated easily and applied to chip-based biosensors without concern about disrupting their function.

Although there is still a long way to go in optimizing the structures and functions of MBs, the future of MBs and their derivatives is bright.

References

1. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308.
2. Whitcombe, D.; et al. Nat. Biotechnol. 1999, 17, 804–807.
3. Zhang, P.; Beck, T.; Tan, W. Angew. Chem., Int. Ed. 2001, 40, 402–405.
4. Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Genet. Anal. Biomol. Eng. 1999, 14, 151–156.
5. Smit, M. L.; et al. Clin. Chem. 2001, 47, 739–744.
6. Trépanier, H.; et al. One-hour Detection of Candida albicans and Candida dubliniensis in Blood Samples Using the Smart Cycler. (Pos ter available at www.smartcycler.com/.)
7. Liu, X.; Tan, W. Anal. Chem. 1999, 71, 5054–5059.
8. Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126–131.