Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with ...an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.
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The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, ...and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient's normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample.
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Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing ...be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA-binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, whereas the preceding and succeeding introns are removed cotranscriptionally.
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► Constitutive splicing occurs at gene loci cotranscriptionally ► Splicing and transcription are uncoupled when polypyrimidine tracts are masked ► Sxl- and PTB-regulated introns are removed in the nucleoplasm after transcript release ► Posttranscriptionally spliced pre-mRNAs tend to cluster around nuclear speckles
Regulators of alternative splicing can delay excision of specific introns until after transcription, without disrupting cotranscriptional splicing of neighboring introns.
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Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterial species that comprises three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus ...subsp. bolletii. These predominantly environmental microorganisms have emerged as life-threatening chronic pulmonary pathogens in both immunocompetent and immunocompromised patients, and their acquisition of macrolide resistance due to the
(41) gene and mutations in the 23S
gene has dramatically impacted patient outcome. However, standard microbiology laboratories typically have limited diagnostic tools to distinguish M. abscessus subspecies, and the testing for macrolide resistance is often not done. Here, we describe the development of a real-time multiplex assay using molecular beacons to establish a robust, rapid, and highly accurate method to both distinguish M. abscessus subspecies and to determine which strains are susceptible to macrolides. We report a bioinformatic approach to identify robust subspecies sequence targets, the design and optimization of six molecular beacons to identify all genotypes, and the development and application of a 2-tube 3-color multiplex assay that can provide clinically significant treatment information in less than 3 h.
The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously ...described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.
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We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR ...assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.
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Mechanism of mRNA Transport in the Nucleus Diana Y. Vargas; Arjun Raj; Salvatore A. E. Marras ...
Proceedings of the National Academy of Sciences - PNAS,
11/2005, Volume:
102, Issue:
47
Journal Article
Peer reviewed
Open access
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.
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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.
The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been ...increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases.
In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients' blood samples.
Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner.
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