Molecular beacons: probes that fluoresce upon hybridization.

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.

Wavelength-shifting molecular beacons.

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.

Multicolor molecular beacons for allele discrimination.

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 beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis.

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.

A DNA damage signal is required for p53 to activate gadd45.

We provide direct evidence that overexpression of p53 is not sufficient for robust p53-dependent activation of the endogenous gadd45 gene. When p53 was induced in TR9-7 cells in the absence of DNA damage, waf1/p21 and mdm2 mRNA levels were increased, but a change in gadd45 mRNA was barely detectable. Activation of the gadd45 gene was observed when camptothecin was added to cells containing p53 in the absence of a further increase in the p53 level. Phosphorylation of p53 at serine 15 and acetylation at lysine 382 were detected after drug treatment. It has been suggested that p53 posttranslational modification is critical during activation. However, inhibition of these modifications by wortmannin was not sufficient to block the transactivation of gadd45. Interestingly, after camptothecin treatment, increased DNase I sensitivity was detected at the gadd45 promoter, suggesting that an undetermined DNA damage signal is involved in inducing chromatin remodeling at the gadd45 promoter while cooperating with p53 to activate gadd45 transcription.

Autocatalytic replication of a recombinant RNA.

We demonstrate that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replicase template. A recombinant RNA was constructed by inserting decaadenylic acid between nucleotides 63 and 64 of MDV-1 (+) RNA, using phage T4 RNA ligase. The insert was located away from regions of the template known to be required for the binding of the replicase and for the initiation of product strand synthesis. To minimize the disruption of template structure, we inserted the heterologous sequence into a hairpin loop on the exterior of the molecule. Q beta replicase copied this recombinant RNA in vitro, and the complementary product strands served as templates for the synthesis of additional copies of the original recombinant RNA. The reaction was therefore autocatalytic and the amount of recombinant RNA increased exponentially. A 300-fold amplification of the recombinant RNA occurred within nine minutes. Insertion of biologically significant RNAs into the MDV-1 RNA sequence should allow them to be replicated autocatalytically.

Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases.

Recent developments in DNA and RNA amplification technology are enabling the design of ultra-sensitive diagnostic assays for infectious diseases. The leading amplification technology is the polymerase chain reaction (PCR). An alternative approach, Q-beta amplification, also promises remarkable speed and precise quantification of assay results.

N-methyl-D-aspartic acid receptor expression in the dorsolateral prefrontal cortex of elderly patients with schizophrenia.

OBJECTIVE: The N-methyl-D-aspartic acid (NMDA) class of glutamate receptors has received attention in the pathophysiology of schizophrenia because of the similarity between some schizophrenic symptoms and symptoms caused by NMDA antagonists. To determine if NMDA receptor abnormalities were present at the mRNA level, expression of NMDA receptor (NR) subunits NR(1), NR(2A), and NR(2B) was measured in specimens from the dorsolateral prefrontal cortex and the occipital cortex of elderly patients with schizophrenia and normal elderly subjects. METHOD: Postmortem specimens from antemortem assessed and diagnosed elderly patients with schizophrenia (N=26) were compared with those from a neuropathologically and neuropsychiatrically normal elderly comparison group (N=13) and from patients with Alzheimer's disease (N=10). The mRNA expression of the NR(1), NR(2A), and NR(2B) subunits and of postsynaptic density 95 (PSD-95), a protein associated with postsynaptic NMDA receptors, was studied with quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: Expression of NR(1) and NR(2A) but not NR(2B) subunits was higher in the dorsolateral prefrontal cortex and the occipital cortex of patients with schizophrenia than in the normal and Alzheimer's disease groups. In contrast, NR(1) expression was significantly lower in the Alzheimer's disease group. Occipital cortex expression of PSD-95 was higher in the schizophrenic subjects and correlated strongly with the expression of NR(2A) and NR(2B) in both cortical regions and with expression of NR(1) in the occipital cortex. These results were not influenced by neuroleptic exposure history, postmortem interval, or age of the subject. CONCLUSIONS: NMDA receptor subunits are abnormally expressed in elderly patients with schizophrenia. The disproportionate expression of the NR(1) and NR(2A) subunits relative to NR(2B) expression may have implications for the pathophysiology of schizophrenia and the sensitivity of schizophrenic patients to glutamate and glutamatergic drugs.

Localization of the Q beta replicase recognition site in MDV-1 RNA.

Fragments of MDV-1 RNA (a small, naturally occurring template for Q beta replicase) that were missing nucleotides at either their 5' end or their 3' end were still able to form a complex with Q beta replicase. By assaying the binding ability of fragments of different length, it was established that the binding site for Q beta replicase is determined by nucleotide sequences that are located near the middle of MDV-1 RNA. Fragments missing nucleotides at their 5' end were able to serve as templates for the synthesis of complementary strands, but fragments missing nucleotides at their 3' end were inactive, indicating that the 3'-terminal region of the template is required for the initiation of RNA synthesis. The nucleotide sequences of both the 3' terminus and the central binding region of MDV-1 (+) RNA are almost identical to sequences at the 3' terminus and at an internal region of Q beta (-) RNA.

Sequencing of pools of nucleic acids on oligonucleotide arrays.

In sequencing-by-hybridization methods, the nucleotide sequence of a nucleic acid is reconstructed by overlapping oligonucleotides capable of hybridizing with the nucleic acid. In their present form, the methods are hardly suitable for sequencing of long nucleic acid molecules because of the occurrence of non-unique overlaps between the oligonucleotides, and similarly to the conventional sequencing methods, it is necessary to obtain an individual molecule. In the method described here, most ambiguities in reconstruction of a sequence from the constituent oligonucleotides are eliminated by preparing on oligonucleotide arrays and separate surveying of the nucleic acid nested partials. This enables longer nucleic acids to be sequenced, and results in a high redundancy of the input data allowing most hybridization errors to be eliminated by algorithmic means. Furthermore, large pools of nucleic acid strands can be sequenced directly, without isolating individual strands.

Amplifiable hybridization probes.

Amplifiable hybridization probes enable the development of extremely sensitive clinical assays. These novel molecules consist of a probe sequence embedded within the sequences of a replicatable RNA. The molecules are first hybridized to target sequences in a conventional manner. The probe-target complexes are then isolated and the probes are released from their targets. The released probes are then amplified by incubation with the RNA-directed RNA polymerase, Q-beta replicase. The replicase copies the probes in a geometrically increasing manner: after each round of copying, the number of RNA molecules is twice the previous number. The doubling process is very rapid, resulting in as many as one billion copies of each molecule in 30 minutes. The amount of RNA that is made is large enough to be measured without using radioisotopes. Theoretically, these assays should be extraordinarily sensitive, since only one probe molecule is required to start the amplification process. In practice the sensitivity of the assays is limited by the presence of non hybridized probes that persist, despite extensive washing of the probe-target hybrids. Currently, the limit of detection is about 10,000 molecules of target. However, replicatable probes are now being prepared that include a "molecular switch", which is a region of the RNA that undergoes a conformational change when the probe sequence hybridizes to its target. Protocols are being developed that link signal generation to the state of this switch. The simplicity and speed of the enzymatic steps that are required facilitate automation of the assays.

Oligonucleotide arrays: new concepts and possibilities.

Advances in solid-phase oligonucleotide synthesis and hybridization techniques have led to an incipient technology based on the use of oligonucleotide arrays. The inclusion of a large number of oligonucleotide probes within a single array greatly reduces the cost of their synthesis and allows thousands of hybridizations to be carried out simultaneously. The range of potential applications of oligonucleotide arrays was expanded by the realization that nucleic acids can be sequenced by hybridizing them to all possible oligonucleotides of a given length. Additional possibilities are offered by novel types of oligonucleotide arrays that are capable of parallel sorting, isolating, and manipulating thousands, and even millions, of nucleic acid species. Fields, such as site-directed mutagenesis, protein engineering, and recombinant DNA technology, would benefit from using these arrays. Further, these approaches could enable the analysis of entire genomes by preparing ordered fragment libraries, and by sequencing complex pools of nucleic acids, in a novel approach that provides long-range sequence information by generating nested nucleic acids and then surveying the oligonucleotides contained in the nested strands. This would allow large diploid genomes to be sequenced directly in a completely automated procedure that does not require fragment cloning or chromosome mapping.

Amplifiable hybridization probes containing a molecular switch.

In order to reduce background signals in Q beta replicase-mediated bioassays, a target-dependent probe amplification strategy has been proposed that utilizes recombinant RNA hybridization probes that contain an inserted molecular switch. A molecular switch is an internal region of the probe that undergoes a conformational change when the probe hybridizes to its target. We investigated whether non-hybridized probes (which cause background signals) could be selectively destroyed by incubating the probe-target hybrids with ribonuclease III, which should cleave the non-hybridized probes and leave the hybridized probes intact. Two problems with this assay design were observed. First, ribonuclease III cleaved probe-target hybrids non-specifically when the target was an RNA, thereby destroying all of the bound probes. And second, the expected conformational change in the molecular switch did not occur when the probes were bound to their targets, apparently because the hairpin stem formed by the molecular switch was too long. Although these results demonstrated that the original assay design could not work, they provided insights that have led to better designs for target-dependent amplification assays. In these assays, the probes will be DNA molecules containing short-stemmed molecular switches. Non-hybridized probes will be selectively destroyed by incubation with a restriction endonuclease.

Structure-independent nucleotide sequence analysis.

Substitution of inosine for granosine in the nucleic acid fragments synthesized for the sequencing of RNA effectively prevents the formation of secondary structures during electrophoretic analysis. Consequently, the mobility of each fragment in the sequencing gel is a strict function of its molecular weight. Inosine substitution should markedly improve the resolution that can be obtained in the sequencing of DNA as well as RNA.

Secondary structure formation during RNA synthesis.

We observed the secondary structures that formed in an RNA molecule during its synthesis. Some of the secondary structures seen in nascent chains were observed to form, then to dissociate in favor of an alternative structure, and then to reform, as chain growth continued. The results show that secondary structures in an RNA molecule are in a state of dynamic equilibrium, and that the extension of a sequence by chain growth, or the reduction of a sequence by processing, may result in significant changes in the secondary structures that are present.

RNA sequencing with radioactive chain-terminating ribonucleotides.

A rapid method for determining nucleotide sequences in RNA is described. It employs the 3'-deoxy analogues of the ribonucleoside triphosphates as specific chain terminators during RNA synthesis. For example, the inclusion of 3'-deoxyuridine 5'-triphosphate in an RNA synthesis reaction in addition to the four usual ribonucleoside triphosphate precursors results in the synthesis of a set of different-length product strands that terminate in a 3'-deoxyuridine that has been incorporated in place of uridine. To sequence an RNA, four separate reactions are run, each employing a different 3'-deoxy terminator. Parallel electrophoretic analysis of the resulting four sets of specifically terminated product chains leads to a direct reading of the nucleotide sequence. We tested this method by sequencing MDV-1 (-) RNA, a molecule that is synthesized in vitro by phage Qbeta replicase. The sequence read from the resulting gels agreed completely with the known sequence of MDV-1 (-) RNA. The bands in some regions of the sequencing gels were unusually close to one another, as has also been observed in other rapid sequencing procedures, making order assignment in these regions very difficult. Because the secondary structure of MDV-1 (-) RNA was known, it was shown that the compression of the bands is due to the persistence of secondary structures during electrophoresis. Thus, structured regions of nucleic acids may introduce difficulties for sequencing techniques that employ the currently available methods of gel electrophoresis.

Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators.

RNA synthesis by T7 RNA polymerase or SP6 RNA polymerase is 100-1000 times more sensitive to the presence of the 3'-deoxyribonucleoside 5'-triphosphate chain terminators than is RNA synthesis by Escherichia coli RNA polymerase or Q beta replicase. These ribonucleotide analogues do not alter the specificity of each polymerase for its own promoters nor do they alter the site at which synthesis is initiated. Transcription by T7 RNA polymerase or SP6 RNA polymerase in the presence of relatively low concentrations of these chain terminators offers a useful route for determining the nucleotide sequence of any DNA segment that is inserted immediately downstream from a homologous bacteriophage promoter. This sequencing procedure was used to explore the effects that different dinucleotides have on the specificity of initiation at two different T7 RNA polymerase promoters.