Peptide Nucleic Acids (PNAs) for Fluorescence in Situ Hybridization (PNA-FISH)
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What is the fluorescence in situ hybridization?
Fluorescence in situ hybridization (FISH) is a technique that does not depend on sequencing. It uses a fluorescently labeled probe, which is either DNA or RNA, with a complementary sequence to the target sequence to hybridize. Epifluorescence, confocal laser scanning microscopy, or flow cytometry can be used to identify or estimate biomass in complex communities and substrates using this technology. In principle, when hybridizing for ribosomal RNA or DNA, the probe molecule will stain any cell that contains rDNA or rRNA. For increased sensitivity (i.e., because of larger copy numbers), ribosomal RNA is usually targeted instead of rDNA (the rRNA gene). Fixation and permeabilization, hybridization, washing to remove unbound probe, and detection/quantification of labeled cells are the four processes that make up a standard FISH method. Permeation of the cells is essential for the penetration of labeled probes, and fixing the samples (e.g., on glass slides, on membrane filters, or in paraffin sections) is required to avoid the destruction of nucleic acids, especially RNA. Probes have been labelled using a variety of approaches. Direct fluorescent labeling is the go-to method due to its low cost, speed, and lack of need for extra sample processing. By using FISH, scientists can see individual fungus on different types of substrates, which helps them learn a lot about biomass and where it's distributed. Cryohistology is the primary method for microscopy, however paraffin histology is also an option. Flow cytometry or fluorescence microscopy in combination with image analysis software allows for cell quantification (FCM-FISH).
What is the PNA-FISH?
PNA-FISH (Peptide Nucleic Acid Fluorescence In Situ Hybridization) is a customized method that integrates the principles of FISH with the distinctive characteristics of peptide nucleic acids (PNAs). This technique is employed for the precise identification and localization of nucleic acid sequences within preserved cells or tissues. PNA is a synthetic analogue of DNA and RNA featuring a backbone made of peptide-like linkages, rendering it more durable and resistant to enzyme destruction. PNAs have great specificity and affinity for binding to complementary DNA or RNA sequences. In PNA-FISH, fluorescently tagged PNA probes are engineered to hybridize with target nucleic acid sequences inside the sample. The binding of the probes is identified by fluorescence microscopy.
PNA probe for FISH. (Mueller P., et al., 2016)
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PNA-FISH for detection of Candida infections
Through the measurement of clinical outcomes such as time to culture clearance, hospital length of stay, and hospital mortality, as well as the median time to Candida species identification and time to targeted therapy, the utility of incorporating the PNA FISH assay with an antimicrobial stewardship intervention in hospitalized patients with candidemia was assessed. Secondary goals included validating the accuracy of the PNA FISH assay and calculating the cost-effectiveness of the test by comparing the expected prices of antifungal drugs (as average wholesale price) before and after the test was implemented (June 26, 2009–September 19, 2010 and September 20, 2010–June 13, 2011). In both cases, the doctor was informed of the Gram stain results by the lab staff after the blood culture was taken. Following the introduction of the PNA FISH test, there was a considerable reduction in the time required for targeted treatment (p = 0.0016). The rate of culture clearance was greater in the group that was treated after the implementation (p = 0.01). The PNA FISH test resulted in a median time to species identification of 0.2 days, while standard techniques took 4 days (p < 0.001). Taking into consideration both the initial investment in the test and the instances where patients were referred to more costly treatments based on the results, we calculated that the PNA FISH test saved around $415 for every patient. A combination of a pharmacy-driven antimicrobial stewardship policy and a PNA FISH test for Candida species identification from yeast-positive blood cultures shortened the duration to targeted antifungal treatment and culture clearance.
The labeling process with the PNA FISH probe. (Heil E L., et al., 2012)
PNA-FISH for detection of bacteria
It can take a long time—approximately 12 to 48 hours—to identify the organisms that cause sepsis-associated bacteremia. These organisms can be mono- or polymicrobial. Laboratory culture processes and rapid bacterial growth rates (up to 5 days) make the process worse. Delays in targeted antimicrobial treatment are linked to longer culture characterization periods, which in turn leads to less than ideal results.
Following the method outlined by Huang‘s team, PNA-FISH-acoustic flow cytometry (PNA-FISH-AFC) allows for the identification of bacteria in blood cultures by combining PNA-FISH with acoustic flow cytometry, specifically targeting eubacterial 16s rRNA. Traditional applications of flow cytometry include blood cell analysis; the acoustic-enhanced flow cytometer concentrates cells into a single stream by means of ultrasonic waves, enabling the assessment of chemical and physical characteristics of target cells. The development of a 15-mer PNA probe coupled to Alexa Fluor 488 allowed visibility, and it was derived from the eubacterial 16S rRNA-targeted PNA-FISH probe. The development of these assays was based on the simulation of blood cultures of E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.
The PNA-FISH-AFC technology outperformed other industry standards including MALDI-TOF and FilmArray in terms of the amount of time it took to detect E. coli, K. pneumoniae, and P. aeruginosa. At a bacterial concentration of 103–104 CFU/mL, E. coli and K. pneumonia took 5–6 hours to be detected, whereas P. aeruginosa took 10 hours. With the present BacTEC system, testing for E. coli and K. pneumonia takes at least 13–14 hours, while testing for P. aeruginosa takes 19–21 hours, which corresponds to a significantly higher bacterial concentration (107–109 CFU/mL). Positive blood cultures may be immediately applied to using this PNA-FISH-AFC technique at an earlier time point and without culture preparation, in reference to existing standards of bacterial identification. Potentially lowering rates of side effects, antimicrobial resistance (AMR), and mortality from pathogenic infections, this expedited bacterial identification allows for the faster administration of the appropriate antimicrobials.
In order to identify Legionella bacteria in polluted water samples, Nácher-Vázquez's group developed a new PNA-FISH technique that does not require the presence of cultured bacteria. The highly conserved 16S rRNA gene sequences of Legionella were aligned and compared with other non-target bacterial strains that may be present in water in order to build the PNA probe. Legionella pneumonophila is thought to be responsible for 97% of all infectious outbreaks. The chosen FISH-PNA probe, LEG22, aims to target the 16S rRNA region between positions 634 and 648 of this subspecies. With a sensitivity and specificity of 100%, the LEG22 probe detected all strains of Legionella in artificially inoculated tap water samples that were contaminated with both Legionella and non-Legionella bacteria. No hybridization of other species was observed. It was only at quantities of at least 101 CFU/L that bacteria could be detected after filtering; at lower concentrations, the detection limit of FISH utilizing microscope equipment prevented the identification of LEG22.
DAPI staining and PNA-FISH were used in combination for detection of staphylococci. (Fazli M., et al., 2014)
PNA-FISH applications in human cell
The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma-globin mRNA in fixed fetal NRBC is investigated. Hemin-induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non-pregnant women and analysed using both slide and flow FISH protocols. Post-chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow-sorted (2 × 106 NBC/sample). Y chromosome–specific PNA FISH was used to confirm that the identified gamma-globin mRNA stained cells were of fetal origin. Flow FISH isolated gamma-globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope. The gamma-globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma-globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies.
The application of chromosome-specific PNA probes on human lymphocytes, amniocytes, isolated blastomeres, and gametes, as well as PNA-FISH tests for in situ chromosomal examinations, has opened new and promising avenues for PNAs in genetic diagnostics. Adapting previously established multi-color PNA-FISH techniques to human spermatozoa has resulted in a novel, rapid technique that has promising applications for the evaluation of aneuploidy in male gametes.
Advantages of PNA-FISH
High specificity: The robust binding affinity of PNAs to their targets results in less background noise and enhanced specificity relative to traditional DNA probes, hence increasing signal detection.
Stability: PNAs exhibit resistance to nuclease degradation, enabling them to preserve integrity during hybridization and washing procedures, hence yielding more dependable tests.
Shorter Hybridization times: PNA-FISH often necessitates shorter incubation periods owing to the rapid binding kinetics of PNAs, hence enhancing efficiency.
No Secondary probes required: PNA-FISH facilitates direct detection, hence streamlining the process, in contrast to conventional FISH, which frequently need supplementary antibodies or probes.
References
- Mueller P., et al., PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei, Experimental Cell Research, 2016, 345(1): 51-59.
- Heil E L., et al., Impact of a rapid peptide nucleic acid fluorescence in situ hybridization assay on treatment of Candida infections, American Journal of Health-System Pharmacy, 2012, 69(21): 1910-1914.
- MacLelland V., et al., Therapeutic and diagnostic applications of antisense peptide nucleic acids, Molecular Therapy-Nucleic Acids, 2023.
- Fazli M., et al., PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples, In Situ Hybridization Protocols, 2014: 261-271.
- Larsen R D., et al., Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization, Prenatal diagnosis, 2003, 23(1): 52-59.
- Pellestor F., et al., PNA–FISH on Human Sperm, Fluorescence in situ Hybridization (FISH) Protocols and Applications, 2010: 283-289.
