Peptide Nucleic Acids (PNAs) for Diagnosis and Detection

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What is the molecular diagnosis and detection?

Molecular diagnosis and detection is a field within medical and biological sciences that involves the examination of biological markers in the genome (DNA), proteome (proteins), and transcriptome (RNA) to identify diseases, infections, or genetic predispositions. This technology highlights the molecular characteristics of cells and tissues, enabling more precise and personalized diagnoses compared to traditional methods.

What is the role of PNAs in diagnosis and detection?

Peptide nucleic acids (PNAs) are increasingly utilized in molecular diagnostics and detection due to their unique properties that enhance selectivity, stability, and versatility. They may be designed to target specific nucleic acid sequences, making them suitable for identifying many conditions, including infectious diseases, genetic disorders, and cancers. PNAs can be integrated into quantitative PCR (qPCR) tests, facilitating the measurement of pathogen burden in clinical specimens. PNA probes have been employed to identify Mycobacterium tuberculosis and many viruses, such as HIV and hepatitis. PNAs can be included into many detection systems, such as fluorescence in situ hybridization (PNA-FISH), PCR-based methods, and microarray technologies, hence enhancing its application in diagnostics.

The unique properties of PNAs lead to less background noise in assays, hence enhancing the clarity and reliability of detection results. This is essential for accurately quantifying low-abundance targets. PNAs can be employed in carrier screening for genetic illnesses, facilitating the early identification of persons at risk of transmitting genetic abnormalities. Utilizing PNA probes in methodologies such as reverse transcription quantitative PCR (RT-qPCR) enables researchers to evaluate gene expression levels in malignancies, facilitating prognosis and therapy choices. PNA probes can be utilized in PNA-FISH to see and locate particular nucleic acid sequences within cells and tissues. This method is essential for analyzing gene expression patterns and chromosomal anomalies in cancer and genetic diseases. PNA can be integrated into microarray systems for high-throughput investigation of gene expression or genetic variation, enabling the simultaneous assessment of thousands of targets in one experiment.

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Use PNA for the detection of nucleic acids in vivo

It is essential in biological research and diagnostics to be able to detect certain nucleic acid sequences. Detecting in intricate biological contexts or even whole cells is a breeze with PNA because to its exceptional hybridization capabilities and metabolic stability. There are two primary approaches to live cell imaging: templated reactions, where a fluorescent product is formed when a target sequence catalyzes its production, and fluorogenic probes, which become more luminous upon duplex formation. Thiazole orange (TO) and its analogues are a highly effective class of dyes for fluorogenic probes. Planarization enhances the fluorescence of these dyes, which is a significant characteristic. An initial demonstration of concept was shown by appending TO to the end of a PNA. Later research indicated that adding TO to the PNA sequence as a base surrogate improved performance. The hybridization process enhances the probes' planarity and fluorescence by a forced intercalation of dye. There was a heightened sensitivity of FIT-PNA probes to a single base pair mismatch close to the TO residue. Additional enhancements were achieved by substituting the aminoethylglycine of the PNA backbone with an ornithine backbone to link the TO. It is worth mentioning that FIT-PNA outperformed a conventional DNA-molecular beacon in both in vitro and cellulo (PNA transfected with sacrificial complementary DNA) tests for the identification of a KRAS oncogenic mutation (SNP). FIT-PNA probes with red-shifted dye (BisQ) successfully sensed SNP in living cells, making them ideal for detection in a cellular setting. The detection of triplex has also been made possible with FIT-PNA probes. Oligomers containing TO may report on triplex formation with single base pair discrimination, much like duplexes, and TO operates as a universal base in a triplex. Using binary probes for a FRET read-out was an additional improvement of FIT-PNA. Shorter oligomers with improved mismatch discrimination can be used with binary probes, which also preserve the unique targeting of longer oligonucleotide stretches. A FIT-PNA probe that could FRET with an NIR-acceptor dye on the neighboring probe was created by combining TO and JO. This allowed for the distinction of a C →U edit in an mRNA of living cells. Colorimetric differentiation between transcript existence and editing status based on emission wavelength is also made possible by the binary probe technology.

A new sensing method for detecting particular DNA target sequences has been developed, which relies on a functionalized microstructured optical fiber-Bragg grating. Covalently attaching a PNA probe to the inner surface of a microstructured fiber—the same surface that had a Bragg grating etched on it earlier—targets a DNA sequence that has a single point mutation that is associated with cystic fibrosis (CF) illness. In accordance with the Watson-Crick pairing, a solution of an oligonucleotide (ON) that corresponds to a tract of the CF gene that contains the altered DNA has been introduced into the fiber capillaries and given the opportunity to hybridize with the fiber surface. Once inside, ON-functionalized gold nanoparticles were utilized in a sandwich-like experiment to create signal amplification. Fluorescence measurements have verified the successful hybridization, and experimental measurements reveal a distinct change in the reflected high order mode of a Bragg grating for a 100 nM DNA solution. It is possible to reuse the sensor because many trials on the same fiber with the same dosage have shown the same modulation trend. Additionally, the sensor's great selectivity was demonstrated by measurements taken using a 100 nM mismatched DNA solution that corresponded to the wild-type gene and had a single nucleotide mutation.

Peptide nucleic acid (PNA) probe for DNA detection. (Candiani A., et al., 2013)

Using PNA for detecting nucleic acid. (Saarbach J., et al., 2019)

Use PNA for detection of RNA

The molecular recognition event, which is usually the hybridization of PNA to target miRNA, is converted into a detectable signal by charge buildup in electrochemical miRNA biosensors. The neutrality of PNA causes a negative charge to accumulate during PNA-RNA hybridization; this charge can be enhanced by adding a reporter unit, such as nanoparticles of gold, iron oxide, silver, or metal/organometallic combinations. A biosensor for miR-21, which is overexpressed in the majority of malignancies, was developed by Kangkamano's group using pyrrolidinyl PNA polypyrrole (acpcPNA) and silver nanofoam (AgNF). In order to quickly and accurately detect the presence of unlabeled miR-21 in blood plasma samples, their developed biosensor could detect electrochemical changes in a current.

The sensor was made by electropolymerizing pyrrole onto an AgNF layer that had been electrodeposited onto a basal Au electrode. After that, the target miRNAs were hybridized by allowing them to drop onto the PNA-immobilized electrode. Using cyclic voltammetry, the signal was measured as the difference in current of the AgNF redox reaction before and after acpcPNA hybridization to target miRNA. We found that the apcPNA-15 mer probe was the optimal PNA probe length, and that the limit of detection dropped as probe concentration increased. It was discovered that the quantity of signal recorded was directly proportional to the quantities of miR-21, and the biosensor could distinguish between single, double, and non-complementary targets. To represent varied miR-21 concentrations in a human model, blood serum samples were taken and diluted. In samples exceeding the 20 fentometer limit, miR-21 could be identified.

Applications of PNA as diagnostic agents. (MacLelland V., et al., 2023)

A PNA chimera consists of the PNA fragment itself, a molecular imaging functional component (such as a radioactive isotope or fluorophore), and a peptide or other surface-bound vector for targeting cell surface transporters or receptors. With weights generally ranging from 4 to 6 kDa, compounds of this kind cannot be considered tiny molecules. Therefore, HPLC/MS, binding experiments on complementary immobilized DNA, and variable-temperature UV/vis spectroscopy are the main tools for characterisation. One possible use for these chimeras is as retention enhancers, which would increase the signal-to-background ratio in imaging. It is common practice to use vectors to target overexpressed receptors, like in the case of WT-4185, which provides initial selectivity for the targeted cell line. The PNA segment would attach to the translated messenger RNA of a gene that is either overexpressed or altered after entering the cytoplasm. It seems that this binding improves retention compared to cells lacking the targeted gene. It is also possible to target overexpressed miRNAs instead of mRNA. Therefore, creating imaging agents of the chimera type has the added advantage of being very adaptable and modular. It is theoretically possible to mix the same PNA fragment with any vector and imaging modality. You may tweak the PNA fragment in this design also if you need to, and it won't effect the way you make the chimera in the end. Furthermore, it has been demonstrated that these structures, despite their size, demonstrate quick biodistribution. For 99mTc labeling to allow single photon emission computed tomography (SPECT), the famous Ac-Gly-ala-Gly-Gly ligand is also included in the design. To reduce the likelihood of interference, tiny linkers were inserted between the various capabilities.

PNA was used as the contrast agent. (Exner R M., et al., 2021)

Mice were pretreated with a HER2-affibody−PNA conjugate. (Exner R M., et al., 2021)

Use PNA in microarray for high-throughput diagnosis/detection

When it comes to gene analysis, PNA microarrays are preferable to DNA chips, but their chemical manufacture is a complex and time-consuming procedure. In order to create the PNA microarray using photolithography, Wu's group created an automated synthesizer. When compared to DNA microarrays, the PNA microarrays created by Yang's group perform better. The researchers in this work synthesized PNA and utilized it to construct high-density PNA microarrays. The results show that these microarrays effectively identified both single and multiple base-mismatches. Due to its immense characteristics, PNA-based probes provide an answer to the limitations of DNA-based microarrays, which include selectivity, sensitivity, and stability in different environments. The advantages of PNA-based devices are their fast speed, accuracy, reproducibility, reusability, and storage capacity. The study then added the intercalator "Hoechst 33258" and incubated it to connect with the PNA-DNA complex after the target DNA had been hybridized. The electrochemical analysis method is used to analyze the hybridization event after incubation.

Diagrammatic representation of PNA-based microarray.(Singh K R B., et al., 2020)

References

1. Candiani A., et al., Label-free DNA biosensor based on a peptide nucleic acid-functionalized microstructured optical fiber-Bragg grating, Journal of biomedical optics, 2013, 18(5): 057004-057004.
2. Saarbach J., et al., Peptide nucleic acid (PNA) and its applications in chemical biology, diagnostics, and therapeutics, Current opinion in chemical biology, 2019, 52: 112-124.
3. MacLelland V., et al., Therapeutic and diagnostic applications of antisense peptide nucleic acids, Molecular Therapy-Nucleic Acids, 2023.
4. Exner R M., et al., Explorations into peptide nucleic acid contrast agents as emerging scaffolds for breakthrough solutions in medical imaging and diagnosis, ACS omega, 2021, 6(43): 28455-28462.
5. Singh K R B., et al., Potential applications of peptide nucleic acid in biomedical domain, Engineering Reports, 2020, 2(9): e12238.