Journal articles: 'PNA Probes'

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Vilaivan, Tirayut. "Fluorogenic PNA probes." Beilstein Journal of Organic Chemistry 14 (January 29, 2018): 253–81. http://dx.doi.org/10.3762/bjoc.14.17.

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Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years.

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Tano, Hanna, Maryam Oroujeni, Anzhelika Vorobyeva, Kristina Westerlund, Yongsheng Liu, Tianqi Xu, Daniel Vasconcelos, Anna Orlova, Amelie Eriksson Karlström, and Vladimir Tolmachev. "Comparative Evaluation of Novel 177Lu-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting." Cancers 13, no. 3 (January 28, 2021): 500. http://dx.doi.org/10.3390/cancers13030500.

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Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe ZHER2:342-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with 177Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [177Lu]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all 177Lu-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe’s size and decreased with an increased number of nucleobases. The shortest PNA probe, [177Lu]Lu-HP16, showed the highest tumor-to-kidney ratio. [177Lu]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.

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Kim, Hyun-Joong, and Byron F. Brehm-Stecher. "Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans." Journal of Clinical Microbiology 53, no. 2 (November 26, 2014): 511–21. http://dx.doi.org/10.1128/jcm.02417-14.

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Candida albicansis an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiatingC. albicansfrom otherCandidaspecies are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescencein situhybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel ofC. albicansand various nontargetCandidaspp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power forC. albicansthan P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification ofC. albicansin clinical and related applications, especially when combined with FCM.

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Oliveira, Alexandre C., Hugo A. L. Filipe, and Luís M. S. Loura. "Fluorescent Probes cis- and trans-Parinaric Acids in Fluid and Gel Lipid Bilayers: A Molecular Dynamics Study." Molecules 28, no. 5 (February 28, 2023): 2241. http://dx.doi.org/10.3390/molecules28052241.

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Fluorescence probes are indispensable tools in biochemical and biophysical membrane studies. Most of them possess extrinsic fluorophores, which often constitute a source of uncertainty and potential perturbation to the host system. In this regard, the few available intrinsically fluorescent membrane probes acquire increased importance. Among them, cis- and trans-parinaric acids (c-PnA and t-PnA, respectively) stand out as probes of membrane order and dynamics. These two compounds are long-chained fatty acids, differing solely in the configurations of two double bonds of their conjugated tetraene fluorophore. In this work, we employed all-atom and coarse-grained molecular dynamics simulations to study the behavior of c-PnA and t-PnA in lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), representative of the liquid disordered and solid ordered lipid phases, respectively. All-atom simulations indicate that the two probes show similar location and orientation in the simulated systems, with the carboxylate facing the water/lipid interface and the tail spanning the membrane leaflet. The two probes establish interactions with the solvent and lipids to a similar degree in POPC. However, the almost linear t-PnA molecules have tighter lipid packing around them, especially in DPPC, where they also interact more with positively charged lipid choline groups. Probably for these reasons, while both probes show similar partition (assessed from computed free energy profiles across bilayers) to POPC, t-PnA clearly partitions more extensively than c-PnA to the gel phase. t-PnA also displays more hindered fluorophore rotation, especially in DPPC. Our results agree very well with experimental fluorescence data from the literature and allow deeper understanding of the behavior of these two reporters of membrane organization.

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Chim, Wilson, Abootaleb Sedighi, Christopher L. Brown, Ralph Pantophlet, and Paul C. H. Li. "Effect of buffer composition on PNA–RNA hybridization studied in the microfluidic microarray chip." Canadian Journal of Chemistry 96, no. 2 (February 2018): 241–47. http://dx.doi.org/10.1139/cjc-2017-0319.

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Herein, we report that peptide nucleic acid sequences (PNAs) have been used as the probe species for detection of RNA and that a microfluidic microarray (MMA) chip is used as the platform for detection of hybridizations between immobilized PNA probes and RNA targets. The RNA targets used are derived from influenza A sequences. This paper discusses the optimization of two probe technologies used for RNA detection and investigates how the composition of the probe buffer and the content of the hybridization solution can influence the overall results. Our data show that the PNA probe is a better choice than the DNA probe when there is low salt in the probe buffer composition. Furthermore, we show that the absence of salt (NaCl) in the hybridization buffer does not hinder the detection of RNA sequences. The results provide evidence that PNA probes are superior to DNA probes in term of sensitivity and adaptability, as PNA immobilization and PNA–RNA hybridization are less affected by salt content in the reaction buffers unlike DNA probes.

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Stender, Henrik, Cletus Kurtzman, Jens J. Hyldig-Nielsen, Ditte Sørensen, Adam Broomer, Kenneth Oliveira, Heather Perry-O'Keefe, Andrew Sage, Barbara Young, and James Coull. "Identification of Dekkera bruxellensis(Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes." Applied and Environmental Microbiology 67, no. 2 (February 1, 2001): 938–41. http://dx.doi.org/10.1128/aem.67.2.938-941.2001.

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ABSTRACT A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification ofBrettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomycesisolates from wine, show that the spoilage organismBrettanomyces belongs to the species D. bruxellensis and that the new method is able to identifyBrettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

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Wilks, Sandra A., and C. William Keevil. "Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms." Applied and Environmental Microbiology 72, no. 8 (August 2006): 5453–62. http://dx.doi.org/10.1128/aem.02918-05.

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ABSTRACT Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.

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Stender, Henrik, Kaare Lund, Kenneth H. Petersen, Ole F. Rasmussen, Poonpilas Hongmanee, Håkan Miörner, and Sven E. Godtfredsen. "Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures." Journal of Clinical Microbiology 37, no. 9 (1999): 2760–65. http://dx.doi.org/10.1128/jcm.37.9.2760-2765.1999.

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TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum, which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC (n = 46) or NTM (n = 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%.

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Mach, Kathleen E., Aniruddha M. Kaushik, Kuangwen Hsieh, Pak Kin Wong, Tza-Huei Wang, and Joseph C. Liao. "Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens." Analyst 144, no. 5 (2019): 1565–74. http://dx.doi.org/10.1039/c8an02194e.

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de Lima, Amanda L. R., Carmelita C. B. Cavalcanti, Mariana C. C. Silva, Patrícia M. G. Paiva, Luana C. B. B. Coelho, Eduardo I. C. Beltrão, and Maria T. dos S. Correia. "Histochemical Evaluation of Human Prostatic Tissues withCratylia mollisSeed Lectin." Journal of Biomedicine and Biotechnology 2010 (2010): 1–6. http://dx.doi.org/10.1155/2010/179817.

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Lectins, proteins which selectively recognize carbohydrates, have been used in histochemistry for the evaluation of changes in glycosylation in processes of cellular differentiation and/or dedifferentiation.Cratylia mollisseed lectins (Cramoll 1,4 and Cramoll 3), conjugated to horseradish peroxidase, were used as histochemical probes in human prostate tissues: normal (NP), hyperplasia (BPH), and prostate carcinoma (PCa). The staining pattern of Con-A and Cramoll 1,4 in BPH was more intense than in NP. These lectins also showed staining differences between BPH and PCa; the latter showing decreased staining intensity with an increased degree of malignancy. PNA and Cramoll 3 stained epithelial cells similarly in all diagnoses although they did present intense staining of PCa glands lumen. Corpora amylacea were not differentially recognized by any of the lectins. Cramoll 1,4 and Cramoll 3 seed lectins present themselves as candidates for histochemical probes for prostate pathologies when compared to commercial lectins such as Con-A and PNA.

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Didion, B. A., and R. Bleher. "261 LABELING SEX-SPECIFIC DNA SEQUENCES IN MAMMALIAN SPERM." Reproduction, Fertility and Development 20, no. 1 (2008): 210. http://dx.doi.org/10.1071/rdv20n1ab261.

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While flow cytometric separation of X- andY-chromosome- bearing sperm has advanced to the point of acceptance in the commercial production of sex-preselected cattle, it is important to continue researching this area to improve efficiencies. For example, the difference in DNA sequence between the X- andY-chromosomes has merit as a foundation for an alternative sperm sexing approach that could enable the complete separation and use of an entire ejaculate. We used synthetic DNA mimics conjugated to a fluorescent dye for in situ detection of Y-chromosomes in metaphase preparations of porcine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are synthetic compounds with higher affinity and stability than conventional DNA probes and are used as specific hybridization probes to complementary DNA. The application of PNA probes was demonstrated previously in telomere analysis studies, and we confirmed their efficacy using a CY3-(CCCTAA)3 PNA to probe bull and boar sperm telomeric sequences. Using male porcine somatic cells and theY-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13-15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation of metaphase chromosomes with the PNA produced a localized signal on theY-chromosome. No signals were present when chromosomes of porcine female somatic cells were incubated with the PNA probes. Because chromosomes occupy non-random territories in all cell nuclei including those in sperm, we expected to find centrally located signals in 50% of fixed boar sperm when these were treated with the same PNA as used for the somatic cells. We found the signals present in 161 of 302 (53.3%) sperm to consist of a single, centrally located, round fluorescent dot in the sperm head. Further research is required to establish the uptake of PNA in live sperm toward evaluation of this approach for sperm sexing.

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Ji, Yuhuan, Yijiang Liu, Wanhong Xia, Alexander Behling, Min Meng, Patrick Bennett, and Laixin Wang. "Importance of probe design for bioanalysis of oligonucleotides using hybridization-based LC-fluorescence assays." Bioanalysis 11, no. 21 (November 2019): 1917–25. http://dx.doi.org/10.4155/bio-2019-0154.

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Aim: The importance of the length and/or structure of fluorescently labeled PNA (peptide nucleic acid) probes for quantitative determination of oligodeoxynucleotides (ODNs) is demonstrated in human plasma using hybridization-based LC-fluorescence assays. The length of the PNA probes impacts the peak shape and chromatographic separation of the resulting PNA/ODN hybridization complexes and affects assay sensitivity, dynamic range and carryover. Methods: For quantitative determination of an 18-mer phosphodiester ODN (DNL1818) in human plasma, an assay utilizing an Atto dye-labeled 12-mer PNA probe provided a linear quantitation range of 0.1–50 ng/ml with excellent accuracy and precision (within -5.3–7.73%). Conclusion: This method provides a convenient method for sensitive and specific quantification of ODNs in biological matrix with limited sample volume and no special extraction.

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Tian, X., A. Chakrabarti, N. Amirkhanov, M. R. Aruva, K. Zhang, C. A. Cardi, S. Lai, M. L. Thakur, and E. Wickstrom. "Receptor-mediated internalization of chelator–PNA–peptide hybridization probes for radioimaging or magnetic resonance imaging of oncogene mRNAs in tumours." Biochemical Society Transactions 35, no. 1 (January 22, 2007): 72–76. http://dx.doi.org/10.1042/bst0350072.

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Early external detection of cancer gene activity might enable early treatment of cancer and might reduce cancer mortality. We hypothesized that oncogene mRNA overexpressed at thousands of copies per malignant cell in a zone of transformed cells could be imaged externally by scintigraphic imaging, PET (positron emission tomography) or MRI (magnetic resonance imaging) with PNA (peptide nucleic acid) hybridization probes that include chelators for metal cations and a cyclized peptide analogue of IGF-1 (insulin-like growth factor 1), D(Cys-Ser-Lys-Cys), to mediate internalization by IGF1R (IGF-1 receptor) overexpressed on cancer cells. We observed that human MCF7 breast cancer cells that overexpress IGF1R efficiently internalized fluorescein–chelator–PNA–D(Cys-Ser-Lys-Cys) to the cytoplasm, but not with D(Cys-Ala-Ala-Cys). Scintigraphic imaging of MCF7 xenografts in immunocompromised mice revealed that CCND1 and MYC [99mTc]chelator–PNA–D(Cys-Ser-Lys-Cys) probes yielded xenograft. PET imaging with [64Cu]chelator–PNA–D(Cys-Ser-Lys-Cys) yielded stronger signals. Scintigraphic imaging of human AsPC1 pancreas cancer xenografts with [99mTc]chelator–KRAS PNA–D(Cys-Ser-Lys-Cys) yielded strong xenograft signals. Stronger xenograft image intensities were obtained by PET imaging of [64Cu]chelator–KRAS PNA–D(Cys-Ser-Lys-Cys). MRI required extension of chelator–polydiamidopropanoate dendrimers from the N-termini of the PNA probes to increase the number of contrast paramagnetic gadolinium (III) cations per probe. These results provide a basis for detection of oncogene activity in tissues from outside the body by hybridization with metal–chelator–PNA–peptides that are selectively internalized by cancer cells.

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Azevedo, N. F., M. J. Vieira, and C. W. Keevil. "Establishment of a continuous model system to study Helicobacter pylori survival in potable water biofilms." Water Science and Technology 47, no. 5 (March 1, 2003): 155–60. http://dx.doi.org/10.2166/wst.2003.0307.

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Close association of the pathogen Helicobacter pylori in drinking water biofilms has been suggested. Using a two-stage water model, the survival and development of the pathogen in potable water biofilms was monitored. Filter sterilized tap water was used as the growth medium and the inoculum consisted of a naturally occurring consortium of microorganisms. Biofilms were generated on removable stainless steel coupons that were placed in the second vessel. Novel technology peptide nucleic acid (PNA) molecular probes were used to detect and locate the pathogen in the biofilms. The PNA-labelled oligonucleotide probes were highly specific, and complementary to the helix 6 region of H. pylori 16S rRNA. The pathogen was tracked in the biofilms using epifluorescence microscopy and episcopic differential interference contrast microscopy. Results show that H. pylori can successfully incorporate within biofilms and its presence was detected for up to five days after inoculation. PNA probes provided an easy and quick way of performing fluorescence in situ hybridisation assays in heterogeneous biofilms.

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Lenaerts, Jeremy, Hilary M. Lappin-Scott, and Jonathan Porter. "Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry." Applied and Environmental Microbiology 73, no. 6 (February 2, 2007): 2020–23. http://dx.doi.org/10.1128/aem.01718-06.

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ABSTRACT Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.

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Worden, Alexandra Z., Sallie W. Chisholm, and Brian J. Binder. "In Situ Hybridization of Prochlorococcusand Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes." Applied and Environmental Microbiology 66, no. 1 (January 1, 2000): 284–89. http://dx.doi.org/10.1128/aem.66.1.284-289.2000.

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ABSTRACT A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA inProchlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions.Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.

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Li, Xu, Eberhard Morgenroth, and Lutgarde Raskin. "Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons." Applied and Environmental Microbiology 74, no. 23 (September 26, 2008): 7297–305. http://dx.doi.org/10.1128/aem.01002-08.

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ABSTRACT The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

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Chandler, Darrell P., Jennie R. Stults, Sharon Cebula, Beatrice L. Schuck, Derek W. Weaver, Kevin K. Anderson, Michael Egholm, and Fred J. Brockman. "Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps." Applied and Environmental Microbiology 66, no. 8 (August 1, 2000): 3438–45. http://dx.doi.org/10.1128/aem.66.8.3438-3445.2000.

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ABSTRACT Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO4, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10−21 M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

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Shigeto, Hajime, Takashi Ohtsuki, Akira Iizuka, Yasuto Akiyama, and Shohei Yamamura. "Imaging analysis of EGFR mutated cancer cells using peptide nucleic acid (PNA)–DNA probes." Analyst 144, no. 15 (2019): 4613–21. http://dx.doi.org/10.1039/c9an00725c.

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Sato, Yusuke, Yuki Takahashi, Takaaki Tanabe, and Seiichi Nishizawa. "Conjugating pyrene onto PNA-based fluorescent probes for improved detection selectivity toward double-stranded siRNA." Organic & Biomolecular Chemistry 18, no. 21 (2020): 4009–13. http://dx.doi.org/10.1039/d0ob00794c.

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Yokoi, Yasuhito, Yugo Kawabuchi, Abdullah Adham Zulmajdi, Reiji Tanaka, Toshiyuki Shibata, Takahiro Muraoka, and Tetsushi Mori. "Cell-Penetrating Peptide–Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium." Molecules 27, no. 24 (December 15, 2022): 8944. http://dx.doi.org/10.3390/molecules27248944.

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Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) –peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP–PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP–PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP–PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP–PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

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Didion, B. A., and R. Bleher. "263 LABELING BOVINE SPERM Y-CHROMOSOME SEQUENCE USING DNA MIMICS." Reproduction, Fertility and Development 21, no. 1 (2009): 229. http://dx.doi.org/10.1071/rdv21n1ab263.

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Flow cytometric separation of X- and Y-chromosome bearing bovine sperm is an accepted technology for use at the commercial level. Nevertheless it is important to continue researching the area of gender-preselected sperm for improved efficiencies. We used a synthetic DNA mimic conjugated to a fluorescent dye for in situ detection of Y chromosomes in metaphase preparations of bovine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are a type of DNA mimic having a higher affinity and stability than conventional DNA probes and are used as hybridization probes to complementary DNA. Using male bovine somatic cells and the Y-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13 to 15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation (~1 h) of metaphase chromosomes with the PNA produced a localized signal on the Y-chromosome. No signals were observed when chromosomes of female bovine somatic cells were incubated with the same PNA probe. Because chromosomes occupy non-random territories in all cell nuclei, including sperm, we proposed to find centrally-located signals in 50% of fixed bovine sperm when treated with the same PNA as used for the somatic cells. As expected, we found the PNA signals present in 50% sperm (23/43) existing as a single, centrally-located, round fluorescent dot in the sperm head. Validation studies were also conducted using bovine sperm previously flow sorted into X or Y populations, and we found the signals in accordance to an expected signal present using the PNA (146/165 or 88.5% with PNA signal in presorted Y sperm heads and 13/174 or 7.5% with PNA signal in presorted X sperm heads).

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Ditmangklo, Boonsong, Jaru Taechalertpaisarn, Khatcharin Siriwong, and Tirayut Vilaivan. "Clickable styryl dyes for fluorescence labeling of pyrrolidinyl PNA probes for the detection of base mutations in DNA." Organic & Biomolecular Chemistry 17, no. 45 (2019): 9712–25. http://dx.doi.org/10.1039/c9ob01492f.

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Charoenpakdee, Chayan, and Tirayut Vilaivan. "Quenching of fluorescently labeled pyrrolidinyl peptide nucleic acid by oligodeoxyguanosine and its application in DNA sensing." Organic & Biomolecular Chemistry 18, no. 30 (2020): 5951–62. http://dx.doi.org/10.1039/d0ob01299h.

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Robertson, Kelly L., Liping Yu, Bruce A. Armitage, A. Javier Lopez, and Linda A. Peteanu. "Fluorescent PNA Probes as Hybridization Labels for Biological RNA†." Biochemistry 45, no. 19 (May 2006): 6066–74. http://dx.doi.org/10.1021/bi052050s.

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Kim, Yong-Tae, Jin Woo Kim, Sung Kee Kim, Goon Ho Joe, and In Seok Hong. "Simultaneous Genotyping of Multiple Somatic Mutations by Using a Clamping PNA and PNA Detection Probes." ChemBioChem 16, no. 2 (December 22, 2014): 209–13. http://dx.doi.org/10.1002/cbic.201402640.

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Manicardi, A., E. Gyssels, R. Corradini, and A. Madder. "Furan-PNA: a mildly inducible irreversible interstrand crosslinking system targeting single and double stranded DNA." Chemical Communications 52, no. 42 (2016): 6930–33. http://dx.doi.org/10.1039/c6cc02062c.

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Kolevzon, N., D. Hashoul, S. Naik, A. Rubinstein, and E. Yavin. "Single point mutation detection in living cancer cells by far-red emitting PNA–FIT probes." Chemical Communications 52, no. 11 (2016): 2405–7. http://dx.doi.org/10.1039/c5cc07502e.

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Thompson, Andrew, Mark Prescott, Noorhan Chelebi, John Smith, Tom Brown, and Günter Schmidt. "Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS." Nucleic Acids Research 35, no. 4 (December 9, 2006): e28-e28. http://dx.doi.org/10.1093/nar/gkl1123.

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Abstract The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

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Zafiropoulos, Demetre, E. Facco, and Lucia Sarchiapone. "Biological dosimetry of ionizing radiation: Evaluation of the dose with cytogenetic methodologies by the construction of calibration curves." International Journal of Modern Physics: Conference Series 44 (January 2016): 1660239. http://dx.doi.org/10.1142/s2010194516602398.

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In case of a radiation accident, it is well known that in the absence of physical dosimetry biological dosimetry based on cytogenetic methods is a unique tool to estimate individual absorbed dose. Moreover, even when physical dosimetry indicates an overexposure, scoring chromosome aberrations (dicentrics and rings) in human peripheral blood lymphocytes (PBLs) at metaphase is presently the most widely used method to confirm dose assessment. The analysis of dicentrics and rings in PBLs after Giemsa staining of metaphase cells is considered the most valid assay for radiation injury. This work shows that applying the fluorescence in situ hybridization (FISH) technique, using telomeric/centromeric peptide nucleic acid (PNA) probes in metaphase chromosomes for radiation dosimetry, could become a fast scoring, reliable and precise method for biological dosimetry after accidental radiation exposures. In both in vitro methods described above, lymphocyte stimulation is needed, and this limits the application in radiation emergency medicine where speed is considered to be a high priority. Using premature chromosome condensation (PCC), irradiated human PBLs (non-stimulated) were fused with mitotic CHO cells, and the yield of excess PCC fragments in Giemsa stained cells was scored. To score dicentrics and rings under PCC conditions, the necessary centromere and telomere detection of the chromosomes was obtained using FISH and specific PNA probes. Of course, a prerequisite for dose assessment in all cases is a dose-effect calibration curve. This work illustrates the various methods used; dose response calibration curves, with 95% confidence limits used to estimate dose uncertainties, have been constructed for conventional metaphase analysis and FISH. We also compare the dose-response curve constructed after scoring of dicentrics and rings using PCC combined with FISH and PNA probes. Also reported are dose response curves showing scored dicentrics and rings per cell, combining PCC of lymphocytes and CHO cells with FISH using PNA probes after 10 h and 24 h after irradiation, and, finally, calibration data of excess PCC fragments (Giemsa) to be used if human blood is available immediately after irradiation or within 24 h.

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Mana, Tanushree, Jayanta Kundu, Hiya Lahiri, Sudipta Bera, Jayeeta Kolay, Surajit Sinha, and Rupa Mukhopadhyay. "Molecularly resolved, label-free nucleic acid sensing at solid–liquid interface using non-ionic DNA analogues." RSC Advances 12, no. 15 (2022): 9263–74. http://dx.doi.org/10.1039/d2ra00386d.

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Improved nucleic acid sensing in terms of single nucleobase mismatch discrimination, as achieved by the surface-confined non-ionic PNA and MO capture probes, is exemplified by single molecule force spectroscopy.

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Stender, Henrik, Adam J. Broomer, Kenneth Oliveira, Heather Perry-O'Keefe, Jens J. Hyldig-Nielsen, Andrew Sage, and James Coull. "Rapid Detection, Identification, and Enumeration ofEscherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization." Applied and Environmental Microbiology 67, no. 1 (January 1, 2001): 142–47. http://dx.doi.org/10.1128/aem.67.1.142-147.2001.

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ABSTRACT A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturableEscherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coliwere detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targetingP. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other thanE. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

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Sato, Takaya, Yusuke Sato, Kenta Iwai, Shusuke Kuge, Seiichi Nishizawa, and Norio Teramae. "Synthetic fluorescent probes capable of selective recognition of 3′-overhanging nucleotides for siRNA delivery imaging." Chemical Communications 51, no. 8 (2015): 1421–24. http://dx.doi.org/10.1039/c4cc08800j.

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Peptide nucleic acid (PNA)–thiazole orange (TO) conjugates are developed as fluorescent probes capable of selective recognition of 3′-overhanging nucleotides of siRNAs for an accurate analysis of the siRNA delivery process.

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Molenaar, C. "Visualizing telomere dynamics in living mammalian cells using PNA probes." EMBO Journal 22, no. 24 (December 15, 2003): 6631–41. http://dx.doi.org/10.1093/emboj/cdg633.

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Marin, Violeta L., and Bruce A. Armitage. "RNA Guanine Quadruplex Invasion by Complementary and Homologous PNA Probes." Journal of the American Chemical Society 127, no. 22 (June 2005): 8032–33. http://dx.doi.org/10.1021/ja051102y.

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Brodyagin, Nikita, Martins Katkevics, Venubabu Kotikam, Christopher A. Ryan, and Eriks Rozners. "Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications." Beilstein Journal of Organic Chemistry 17 (July 19, 2021): 1641–88. http://dx.doi.org/10.3762/bjoc.17.116.

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Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA’s chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA’s binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA’s binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.

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De, Arpita, Serhiy Souchelnytskyi, Albert van den Berg, and Edwin T. Carlen. "Peptide Nucleic Acid (PNA)–DNA Duplexes: Comparison of Hybridization Affinity between Vertically and Horizontally Tethered PNA Probes." ACS Applied Materials & Interfaces 5, no. 11 (May 20, 2013): 4607–12. http://dx.doi.org/10.1021/am4011429.

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Sabale, Pramod M., Uddhav B. Ambi, and Seergazhi G. Srivatsan. "Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs." ACS Omega 3, no. 11 (November 12, 2018): 15343–52. http://dx.doi.org/10.1021/acsomega.8b02550.

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Ghosh, Srabani, Sourav Mishra, Trambaki Banerjee, and Rupa Mukhopadhyay. "Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes via Ionic Regulation." Langmuir 29, no. 10 (March 2013): 3370–79. http://dx.doi.org/10.1021/la400125x.

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Köhler, Olaf, Dilip V. Jarikote, Ishwar Singh, Virinder S. Parmar, Elmar Weinhold, and Oliver Seitz. "Forced intercalation as a tool in gene diagnostics and in studying DNA–protein interactions." Pure and Applied Chemistry 77, no. 1 (January 1, 2005): 327–38. http://dx.doi.org/10.1351/pac200577010327.

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Aromatic and heteroaromatic groups that are forced to intercalate at specific positions in DNA are versatile probes of DNA–DNA and DNA–protein recognition. Fluorescent nucleobases are of value since they are able to report on localized alterations of DNA duplex structure. However, the fluorescence of the vast majority of base surrogates becomes quenched upon intercalation in DNA. Peptide nucleic acid (PNA)-based probes are presented in which the intercalator dye thiazole orange (TO) serves as a fluorescent base surrogate. In these probes, fluorescence increases (5–60-fold) upon hybridization. PNA-bearing TO as fluorescent base surrogate could hence prove useful in real-time polymerase chain reaction (PCR) applications and in live cell analysis. Forced intercalation of aromatic polycycles can help to explore the binding mechanism of DNA-modifying enzymes. We discuss studies of DNA-methyltransferases (MTases) which commence methylation of nucleobases in DNA by flipping the target nucleotide completely out of the helix. A method for probing the base-flipping mechanism is suggested. It draws upon the observation that large hydrophobic base surrogates in the face of the swung-out base can enhance the DNA-enzyme binding affinity possibly by disrupting target base-stacking and stabilizing the apparent abasic site.

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Bethge, Lucas, Dilip Venkatrao Jarikote, and Oliver Seitz. "New cyanine dyes as base surrogates in PNA: Forced intercalation probes (FIT-probes) for homogeneous SNP detection." Bioorganic & Medicinal Chemistry 16, no. 1 (January 2008): 114–25. http://dx.doi.org/10.1016/j.bmc.2006.12.044.

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Jarczewska, Marta, Wiktor Bojarski, Aleksandra Majewska, Magdalena Mieczkowska, Marcin Drozd, Robert Ziolkowski, and Elżbieta Malinowska. "Application of Single-Stranded DNA and PNA Probes for Electrochemical Detection of miRNA 141." ECS Meeting Abstracts MA2022-01, no. 53 (July 7, 2022): 2239. http://dx.doi.org/10.1149/ma2022-01532239mtgabs.

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miRNA are known as short RNA molecules that usually consist of 18 – 25 nucleotides and are considered as posttranscriptional regulators of gene expression occurring in plants and animals [1]. Over 60% of all genes which encode proteins are controlled by miRNA [2]. miRNA is engaged in various processes including differentiation, proliferation and apoptosis as well as inflammation. Hence, they might be considered as potential biomarkers of diseases such as cancer, neurodegenerative and cardiovascular [3]. A huge challenge is the actual detection of miRNA because of their short length, low concentration and matrix interferences. The miRNA analysis can be performed with the following techniques such as real-time PCR, northern blotting and microarray [4-6]. However, the above mentioned techniques are rather sophisticated and are costly. An alternative could be the application of electrochemical techniques which are characterized with low detection limits, possibility of miniaturization and high sensitivity. In such a case miRNA could be detected using biosensors containing receptor layers formed of single-stranded DNA or PNA. Herein, we present the studies on the development of ssDNA/PNA – based receptor layers for the electrochemical analysis of miRNA 141, which is a biomarker of pancreatic cancer. Various receptor layers were tested including unlabelled ssDNA, methylene blue-labelled DNA as well as PNA-based. The formation of ssDNA/PNA receptor layer along with its binding to miRNA was confirmed using QCM and SPR techniques. The electrochemical studies revealed that it was not possible to obtain low detection limits for miRNA when MB-labelled ssDNA or PNA molecules were applied. On the contrary, detection limit of 10-4 nM was reached with the application of unlabelled ssDNA molecules. The further analysis showed the possibility of detection of point mutation in ssDNA or miRNA sequence as well as miRNA detection in spiked serum sample. Bibliography: [1] D. P. Bartel, Cell 116 (2004) 281–297 [2] A. Krek, D. Grun, M. N. Poy, R. Wolf, L. Rosenberg, E. J. Epstein, P. MacMenamin, I. da Piedade, K. C. Gunsalus, M. Stoffel, N. Rajewsky, N. Nat. Genet. 37 (2005) 495−500. [3] H. Hwang, J. T. Mendell, Br. J. Cancer 94 (2006) 776–780. [4] C. Chen, D. A. Ridzon, A. J. Broomer, Z. Zhou, D. H. Lee, J. T. Nguyen, M. Barbisin, N. L. Xu, V. R. Mahuvakar, M. R. Andersen, K. Q. Lao, K. J. Livak, K. J.Guegler, Nucleic Acids Res. 33 (2005) e179 [5] A. Valoczi, C. Hornyik, N. Varga, J. Burgyan, S. Kauppinen, Z. Havelda, Nucleic Acids Res. 32 (2004) e175 [6] A. M. Krichevsky, K. S. King, C. P. Donahue, K. Khrapko, K. S.Kosik, RNA 9 (2003) 1274−1281.

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Bonvicini, Francesca, Claudia Filippone, Elisabetta Manaresi, Giovanna Angela Gentilomi, Marialuisa Zerbini, Monica Musiani, and Giorgio Gallinella. "Peptide Nucleic Acid–Based In Situ Hybridization Assay for Detection of Parvovirus B19 Nucleic Acids." Clinical Chemistry 52, no. 6 (June 1, 2006): 973–78. http://dx.doi.org/10.1373/clinchem.2005.064741.

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Abstract Background: Peptide nucleic acid (PNA) molecules are known to bind complementary nucleic acid sequences with a much stronger affinity and with more stable binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic acids by in situ hybridization assay (ISH) and/or PCR, as an experimental model to develop an ISH assay that uses biotinylated PNA probes to detect viral genome in clinical specimens. Methods: We first optimized the PNA-ISH assay on B19-infected and mock-infected UT-7/EpoS1 cells and then tested the assay on archival B19 specimens and on consecutive specimens. All data were compared with data obtained with a standardized DNA-based ISH assay and confirmed by a PCR-ELISA. Results: PNA-ISH detected B19 genome in a higher number of B19-infected UT-7/EpoS1 cells and with a more defined localization of viral nucleic acids than the standardized DNA-ISH assay. Moreover, PNA-ISH was able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH detected more positive samples than DNA-ISH when consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results. Conclusions: The PNA-ISH assay had sensitivity and specificity comparable to a PCR assay and was more practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic test for the detection of viral nucleic acids in clinical specimens.

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De, Arpita, Serhiy Souchelnytskyi, Albert van den Berg, and Edwin T. Carlen. "Correction to Peptide Nucleic Acid (PNA)-DNA Duplexes: Comparison of Hybridization Affinity between Vertically and Horizontally Tethered PNA Probes." ACS Applied Materials & Interfaces 5, no. 15 (July 25, 2013): 7659. http://dx.doi.org/10.1021/am402515h.

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Nácher-Vázquez, Montserrat, Ana Barbosa, Inês Armelim, Andreia Sofia Azevedo, Gonçalo Nieto Almeida, Cristina Pizarro, Nuno Filipe Azevedo, Carina Almeida, and Laura Cerqueira. "Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples." Microorganisms 10, no. 7 (July 13, 2022): 1409. http://dx.doi.org/10.3390/microorganisms10071409.

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Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of Legionella.

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Fortunati, Simone, Andrea Rozzi, Federica Curti, Marco Giannetto, Roberto Corradini, and Maria Careri. "Single-Walled Carbon Nanotubes as Enhancing Substrates for PNA-Based Amperometric Genosensors." Sensors 19, no. 3 (January 30, 2019): 588. http://dx.doi.org/10.3390/s19030588.

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A new amperometric sandwich-format genosensor has been implemented on single-walled carbon nanotubes screen printed electrodes (SWCNT-SPEs) and compared in terms of performance with analogous genoassays developed using the same methodology on non-nanostructured glassy carbon platforms (GC-SPE). The working principle of the genosensors is based on the covalent immobilization of Peptide Nucleic Acid (PNA) capture probes (CP) on the electrode surface, carried out through the carboxylic functions present on SWCNT-SPEs (carboxylated SWCNT) or electrochemically induced on GC-SPEs. The sequence of the CP was complementary to a 20-mer portion of the target DNA; a second biotin-tagged PNA signalling probe (SP), with sequence complementary to a different contiguous portion of the target DNA, was used to obtain a sandwich hybrid with an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). Comparison of the responses obtained from the SWCNT-SPEs with those produced from the non-nanostructured substrates evidenced the remarkable enhancement effect given by the nanostructured electrode platforms, achieved both in terms of loading capability of PNA probes and amplification of the electron transfer phenomena exploited for the signal transduction, giving rise to more than four-fold higher sensitivity when using SWCNT-SPEs. The nanostructured substrate allowed to reach limit of detection (LOD) of 71 pM and limit of quantitation (LOQ) of 256 pM, while the corresponding values obtained with GC-SPEs were 430 pM and 1.43 nM, respectively.

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J. W. A. Boei, S. Vermeulen, A. T., J. "Analysis of radiation-induced chromosomal aberrations using telomeric and centromeric PNA probes." International Journal of Radiation Biology 76, no. 2 (January 2000): 163–67. http://dx.doi.org/10.1080/095530000138817.

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Protozanova, Ekaterina, Ting Wang, Kyoko Uehara, Misha Safranovitch, Rhea Mahabir, Jess Shen, Niru Chennagiri, Douglas B. Cameron, and Rudolf Gilmanshin. "Binding Specificity of Multi-Labeled PNA Probes Studied by Single Molecule Mapping." Biophysical Journal 96, no. 3 (February 2009): 25a. http://dx.doi.org/10.1016/j.bpj.2008.12.017.

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Tilsner, Jens, and Cristina Flors. "FIT for Purpose: PNA-Based Probes Enable mRNA Imaging in Living Cells." ChemBioChem 12, no. 7 (April 4, 2011): 1007–9. http://dx.doi.org/10.1002/cbic.201100139.

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Lee, En Ting Tabitha, Yusuke Sato, and Seiichi Nishizawa. "Small molecule–PNA oligomer conjugates for rRNA A-site at neutral pH for FID assays." Chemical Communications 56, no. 95 (2020): 14976–79. http://dx.doi.org/10.1039/d0cc06084d.

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Journal articles on the topic 'PNA Probes'

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