We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.

Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogenous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore. We found that their hairpin conformation enables the use of a wide variety of differently colored fluorophores. Using several molecular beacons, each designed to recognize a different target and each labeled with a different fluorophore, we demonstrate that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. A comparison of "hairpin probes" with corresponding "linear probes" confirms that the presence of the hairpin stem in molecular beacons significantly enhances their specificity.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched f luorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their f luorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers, including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.

SummaryThe ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real-time RT-PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.

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We developed a new approach to DNA sequence analysis that uses fluorogenic reporter molecules--molecular beacons--and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA. A set of overlapping molecular beacons was used to analyze an 81-bp region of the Mycobacterium tuberculosis rpoB gene for mutations that confer resistance to the antibiotic rifampin. In a blinded study of 52 rifampin-resistant and 23 rifampin-susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy.

The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.gene expression ͉ live cell imaging ͉ mRNA export ͉ nuclear viscosity A fter mRNAs are synthesized, processed, and become associated with a number of different proteins at the transcription site, they are released into the nucleoplasm (1). The mechanism by which these large mRNA-protein (mRNP) complexes then move through dense nucleoplasm to reach the nuclear pores has been the subject of intense study and speculation (2, 3). Early workers proposed that mRNP complexes are transferred along a chain of receptors until they reach a nuclear pore, expending metabolic energy in the process (4). This solid-state transport model is supported by observations made in fixed nuclei that show some transcripts distributed along tracks that originate from the locus of the parent gene (5, 6). A second theory, called the ''gene-gating'' hypothesis, proposes that active genes are situated near the nuclear periphery and that mRNAs exit the nucleus through the nearest pores (7). This idea is supported by observations that certain mRNAs exit from one side of the nucleus (8) and that, in yeast, many transcriptionally active gene loci are located near the nuclear periphery (9). By contrast, a number of other studies have found that mRNP complexes move quite freely within the nucleus (10-16). This view is supported by studies of the distribution of newly synthesized Balbiani ring RNA in the salivary gland cells of insects (11), fluorescence recovery after photobleaching and fluorescence correlation spectroscopy studies of probes that bind to the poly(A) tails of mRNAs (12-15), and from single-particle analysis of mRNP complexes bound to GFP-linked proteins (16).Although the latter studies found that mRNP complexes are able to diffuse within the nuclear matrix, there was a paradoxical active transport component to their motility, because both a reduction in temperature and ATP depletion curtailed the mobility of the complexes (14-16). To better understand the nature of mRNP mobility, we have developed a system of fluorogenic probes and mRNA constructs that allo...