Lighting the way

With the completion of the human genome project, gene analysis has become a critical technique for biomedical research and development and biomedical diagnosis. One recently developed fluorescent DNA probe, the molecular beacon (MB), offers unique advantages for ultrasensitive gene analysis. MBs are novel hairpin-shaped nucleic-acid probes that emit fluorescence when hybridized with their complementary DNA/RNA. Compared with conventional linear DNA probes, MBs offer ultrahigh sensitivity, better specificity due to their unique stem-and-loop structures, and the possibility of "detection without separation."

Our group has developed a variety of applications for MBs in gene analysis.^1,2 Examples include a nanowell array methodology for the analysis of ultrasmall amounts of DNA and RNA (fewer than 10 molecules) with a sample size in each well as small as 28 fL; a single molecule imaging technique to study single DNA molecule hybridization and for ultrasensitive gene identification capability with only one DNA target; and real-time imaging for single-cell mRNA determination using MBs and a laser-induced fluorescence microscopy. These recent advancements in ultrasensitive DNA/RNA analysis using MB probes provide a variety of powerful tools for genomic studies, molecular diagnoses of diseases, and new drug developments.

MBs can enable sensitive, selective, quantitative, and easy analysis of DNA/RNA molecules in homogeneous solutions at the liquid-solid interface and inside living cell specimens. The MB has a stem-loop structure (see figure 1). Its signal transduction mechanism for molecular recognition is based on fluorescence energy transfer. The loop portion of an MB 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 MB's stem, which has five to seven base pairs. A fluorescent moiety is covalently attached to the end of one arm, and a quenching moiety is covalently attached to the other end. The fluorescent dye serves as an energy donor, and the nonfluorescent quencher plays the role of an acceptor. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched. Thus, the probe is unable to fluoresce.

When the probe encounters a target molecule, the loop forms a hybrid that is longer and more stable than the stem. The MB undergoes a spontaneous conformational reorganization that forces the stem apart. The fluorophore and the quencher are moved away from each other, which leads to the restoration of fluorescence. Unhybridized molecular beacons do not fluoresce; thus, it is not necessary to remove them to observe hybridized probes.

Molecular beacon probes have a few important advantages compared with conventional linear DNA fluorescent probes. They are highly sensitive. The built-in signal transduction mechanism allows MBs to act as gene probes with a signal-to-background ratio of better than 200-fold. The beacons also have excellent specificity due to their stem-and-loop structure, which enables MBs to differentiate between DNA probes with a single mismatched base. Since MBs are nonfluorescent when they do not hybridize with their targets, they can be used in situations in which it is either impossible or undesirable to isolate the probe-target hybrids from an excess of the unhybridized probes, for example in detection of mRNAs within living cells or in real-time monitoring of polymerase chain reactions in sealed tubes.

The usefulness of "detection without separation" for these applications cannot be overemphasized. This feature enables the synthesis of nucleic acids to be monitored as the detection is occurring, either in sealed tubes or in living specimen, without additional manipulations.

Our group has developed an MB-based microwell array methodology using laser-induced fluorescence imaging for detection of DNA/RNA molecules. We use a lithographic and wet-chemical-etching technique to fabricate the microwell array. Each 20 X 20 array includes 400 wells, each of which is 6 µm in diameter and 1 µm deep. The volume of each well is about 28 fL. Compared with traditional DNA microarray techniques, the ultrasmall volume involved in our approach reduces the costs associated with reagents and sampling, as well as analysis time.

The instrumental apparatus mainly consists of an inverted Olympus microscope, an intensified-charge-coupled-device (ICCD) detector, an argon ion (Ar⁺) laser (Coherent Inc.; Santa Clara, CA) operating at 514 nm, and a microwell array positioned on the microscope stage. For demonstration purposes, we labeled the MBs with tetramethylrhodamine (TAMRA) as the fluorophore and Dabcyl as the quencher.

For the studies, we mounted the detector on the top port of the microscope and collected fluorescence via a 60X, 0.7NA microscope objective, using an exposure time of 1 s. A dichroic mirror transmitted the fluorescence signal from the hybrid of the molecular beacon and its complementary target to the ICCD. To specifically monitor the fluorescence signal, we placed a bandpass filter centered at 580 nm in front of the optical detector. The laser excitation power was optimized to maximize the sensitivity and minimize the photobleaching.

One of the challenges in this process has been filling the femtoliter wells with liquid. We solved the problem by adding a surfactant reagent 1% Triton X-100 to the solution (the final concentration of the surfactant was 1.0 X 10^-4 M) before liquid filling. Our control experiments showed that the surfactant did not affect MB structure/reactivity.

We used the setup to test gene analysis based on the array of wells. The detection limit with a dye is nine Rhodamin 6G molecules in a 28 fL well. The concentration detection limit of MB probes for a DNA target is around 3.0 nM, which corresponds to 50 molecules in a 28 fL well. Thus, 50 DNA copies can be detected in each well. We have also monitored hybridization of the MB and its complementary oligonucleotide target in the arrayed wells. Consecutive images were taken as the hybridization proceeded. Figure 2 shows that more and more MBs were hybridized with their targets over time, resulting in the new generation of fluorescence signals.

This sensitive and accurate detection method also has been used for the analysis of specific rate Y-actin mRNA sequences amplified by the polymerase chain reaction. Detection of 675 mRNA molecules has been achieved in each well. If we use micromanipulation and microinjection techniques, we can apply the imaging system for MB arrays, acquiring parallel genetic information for multiple analytes on a single cell level.

To perform ultrasensitive gene analysis at a solid/liquid interface, we designed and immobilized biotinlyted MB molecules on the surface of a quartz slide through biotin-avidin interaction. We were able to image single MB molecules and to study the reaction kinetics and dynamics of DNA hybridization on a single molecule basis. We also prepared a single molecular array. We controlled the density of the MB immobilization so that it was low enough that individual MB molecules would be shown in the fluorescence image. The single molecule array is intended for ultrasensitive analysis of genes and for stochastic biosensors for biophotonics applications.

We performed laser induced fluorescence imaging by exciting the immobilized MBs with an evanescent wave field produced at the silica-water interface. We passed the beam from an Ar⁺ laser (Melles Griot; Carlsbad, CA) operating at 514 nm through a λ/4 filter, then used mirrors to direct it through a quartz prism mounted above the objective of an inverted microscope. The sample chamber, consisting of a quartz slide and a cover slip attached by glycerol, was set between the prism and the microscope objective. We adjusted the angles of incidence of the laser beam such that total internal reflection took place at the interface of the quartz slide and the sample solution. A 100X, 1.35-NA oil-immersion objective collected the emission signal and directed it to an ICCD fitted with long-pass and band-pass filters to select the desired fluorescence signal (see figure 3). The single MB molecule array presents simultaneous analysis of tens of individual DNA targets and shows that different molecular beacons have different hybridization kinetics with their cDNA.

In order for us to understand the basic cell biological and development processes, we must obtain knowledge of the subcellular localization and cellular transport pathway of mRNAs. The small amount of mRNA molecules (thousands of copies) within a cell requires ultrasensitive probes for detection. Molecular beacons provide a unique tool to study the spatial and temporal distribution of mRNA species in living cells.

The imaging system setup we used in our experiments was similar to the one used for DNA/RNA analysis in well arrays. After microinjection of MBs into the cytoplasm of single, living kangaroo-rat kidney cells, we used a series of laser-induced fluorescence measurements to monitor the MB hybridization with β-action mRNA. We carried out strict control experiments to confirm the specific hybridization of MB and mRNA. The imaging and localization of MB/mRNA interactions open the possibility to study RNA processing, trafficking, and folding in the living cells.

MBs provide a powerful tool for gene analysis. Using two fluorophores to label the MBs, researchers will be able to quantify gene analysis either in solution, at an interface, or inside living specimens. Molecular beacons have excellent specificity with single-base mismatch identification capability, so they promise to be useful for mutation detection in single living cells. These unique properties of MBs will open the possibility for nucleic-acid research and for molecular diagnosis of diseases in the near future. oe

Acknowledgments

This work is supported by the NSF.

References

1. Xiaohong Fang, Jianwei Jeffery Li, John Perlette, Weihong Tan and Kemin Wang, Analytical Chemistry, 72[23], 747A-753A, 2000.

2. Peng Zhang, Terry Beck and Weihong Tan, Design a molecular beacon with two dye molecules, Angewandte Chemie International Edition, 2001, 40, 402-405.

Genomic information determines the form and function of an organism. Genes, the fundamental functional units of the genome, encode proteins that play crucial roles in virtually all biological processes. Genomic DNA exists as a double-stranded polymer containing four bases (A, T, C, and G) tethered to a sugar-phosphate backbone. A interacts with T, and C interacts with G through hydrogen binding. Gene expression is the process by which messenger RNA (mRNA), and eventually proteins, are produced from the DNA template of each gene.

The order of bases along the helix is known as the primary sequence of the DNA. Primary sequences of the small genomes of several viruses and bacteria have been determined. With the ongoing revolution brought by the human genome project, the focus will change from sequencing and identifying genes to analysis and understanding of human biology and disorders. Thus, the development of highly sensitive and specific DNA/RNA detection methods is essential for gene diagnosis.