Molecules

Editorial

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4 pages, 378 KiB

Open AccessEditorial

Peptide Nucleic Acids: Applications in Biomedical Sciences

by Eylon Yavin

Molecules 2020, 25(15), 3317; https://doi.org/10.3390/molecules25153317 - 22 Jul 2020

Cited by 9 | Viewed by 2661

Abstract

The DNA mimic, PNA (peptide nucleic acid), has been with us now for almost 3 decades [...] Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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Research

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10 pages, 1631 KiB

Open AccessArticle

l-DNA-Based Catalytic Hairpin Assembly Circuit

by Adam M. Kabza and Jonathan T. Sczepanski

Molecules 2020, 25(4), 947; https://doi.org/10.3390/molecules25040947 - 20 Feb 2020

Cited by 17 | Viewed by 4777

Abstract

Isothermal, enzyme-free amplification methods based on DNA strand-displacement reactions show great promise for applications in biosensing and disease diagnostics but operating such systems within biological environments remains extremely challenging due to the susceptibility of DNA to nuclease degradation. Here, we report a catalytic [...] Read more.

Isothermal, enzyme-free amplification methods based on DNA strand-displacement reactions show great promise for applications in biosensing and disease diagnostics but operating such systems within biological environments remains extremely challenging due to the susceptibility of DNA to nuclease degradation. Here, we report a catalytic hairpin assembly (CHA) circuit constructed from nuclease-resistant l-DNA that is capable of unimpeded signal amplification in the presence of 10% fetal bovine serum (FBS). The superior biostability of the l-DNA CHA circuit relative to its native d-DNA counterpart was clearly demonstrated through a direct comparison of the two systems (d versus l) under various conditions. Importantly, we show that the l-CHA circuit can be sequence-specifically interfaced with an endogenous d-nucleic acid biomarker via an achiral peptide nucleic acid (PNA) intermediary, enabling catalytic detection of the target in FBS. Overall, this work establishes a blueprint for the detection of low-abundance nucleic acids in harsh biological environments and provides further impetus for the construction of DNA nanotechnology using l-oligonucleotides. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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20 pages, 4179 KiB

Open AccessArticle

SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens

by Kenji Takagi, Tenko Hayashi, Shinjiro Sawada, Miku Okazaki, Sakiko Hori, Katsuya Ogata, Nobuo Kato, Yasuhito Ebara and Kunihiro Kaihatsu

Molecules 2020, 25(4), 769; https://doi.org/10.3390/molecules25040769 - 11 Feb 2020

Cited by 3 | Viewed by 3681

Abstract

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools [...] Read more.

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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14 pages, 1433 KiB

Open AccessArticle

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

by Alan Ann Lerk Ong, Jiazi Tan, Malini Bhadra, Clément Dezanet, Kiran M. Patil, Mei Sian Chong, Ryszard Kierzek, Jean-Luc Decout, Xavier Roca and Gang Chen

Molecules 2019, 24(16), 3020; https://doi.org/10.3390/molecules24163020 - 20 Aug 2019

Cited by 17 | Viewed by 5293

Abstract

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of [...] Read more.

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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Review

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25 pages, 5129 KiB

Open AccessFeature PaperReview

PNA-Based MicroRNA Detection Methodologies

by Enrico Cadoni, Alex Manicardi and Annemieke Madder

Molecules 2020, 25(6), 1296; https://doi.org/10.3390/molecules25061296 - 12 Mar 2020

Cited by 29 | Viewed by 7043

Abstract

MicroRNAs (miRNAs or miRs) are small noncoding RNAs involved in the fine regulation of post-transcriptional processes in the cell. The physiological levels of these short (20–22-mer) oligonucleotides are important for the homeostasis of the organism, and therefore dysregulation can lead to the onset [...] Read more.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs involved in the fine regulation of post-transcriptional processes in the cell. The physiological levels of these short (20–22-mer) oligonucleotides are important for the homeostasis of the organism, and therefore dysregulation can lead to the onset of cancer and other pathologies. Their importance as biomarkers is constantly growing and, in this context, detection methods based on the hybridization to peptide nucleic acids (PNAs) are gaining their place in the spotlight. After a brief overview of their biogenesis, this review will discuss the significance of targeting miR, providing a wide range of PNA-based approaches to detect them at biologically significant concentrations, based on electrochemical, fluorescence and colorimetric assays. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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13 pages, 593 KiB

Open AccessReview

PNA Clamping in Nucleic Acid Amplification Protocols to Detect Single Nucleotide Mutations Related to Cancer

by Munira F. Fouz and Daniel H. Appella

Molecules 2020, 25(4), 786; https://doi.org/10.3390/molecules25040786 - 12 Feb 2020

Cited by 22 | Viewed by 6343

Abstract

This review describes the application of peptide nucleic acids (PNAs) as clamps that prevent nucleic acid amplification of wild-type DNA so that DNA with mutations may be observed. These methods are useful to detect single-nucleotide polymorphisms (SNPs) in cases where there is a [...] Read more.

This review describes the application of peptide nucleic acids (PNAs) as clamps that prevent nucleic acid amplification of wild-type DNA so that DNA with mutations may be observed. These methods are useful to detect single-nucleotide polymorphisms (SNPs) in cases where there is a small amount of mutated DNA relative to the amount of normal (unmutated/wild-type) DNA. Detecting SNPs arising from mutated DNA can be useful to diagnose various genetic diseases, and is especially important in cancer diagnostics for early detection, proper diagnosis, and monitoring of disease progression. Most examples use PNA clamps to inhibit PCR amplification of wild-type DNA to identify the presence of mutated DNA associated with various types of cancer. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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21 pages, 1115 KiB

Open AccessReview

Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair

by Nicholas G. Economos, Stanley Oyaghire, Elias Quijano, Adele S. Ricciardi, W. Mark Saltzman and Peter M. Glazer

Molecules 2020, 25(3), 735; https://doi.org/10.3390/molecules25030735 - 8 Feb 2020

Cited by 46 | Viewed by 8332

Abstract

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct [...] Read more.

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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22 pages, 2602 KiB

Open AccessReview

Antibacterial Peptide Nucleic Acids—Facts and Perspectives

by Monika Wojciechowska, Marcin Równicki, Adam Mieczkowski, Joanna Miszkiewicz and Joanna Trylska

Molecules 2020, 25(3), 559; https://doi.org/10.3390/molecules25030559 - 28 Jan 2020

Cited by 50 | Viewed by 8719

Abstract

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common [...] Read more.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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Other

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12 pages, 1466 KiB

Open AccessBrief Report

A Peptide Nucleic Acid (PNA) Masking the miR-145-5p Binding Site of the 3′UTR of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNA Enhances CFTR Expression in Calu-3 Cells

by Shaiq Sultan, Andrea Rozzi, Jessica Gasparello, Alex Manicardi, Roberto Corradini, Chiara Papi, Alessia Finotti, Ilaria Lampronti, Eva Reali, Giulio Cabrini, Roberto Gambari and Monica Borgatti

Molecules 2020, 25(7), 1677; https://doi.org/10.3390/molecules25071677 - 5 Apr 2020

Cited by 19 | Viewed by 3760

Abstract

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In [...] Read more.

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3′UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3′UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content. Full article

(This article belongs to the Special Issue Peptide Nucleic Acids: Applications in Biomedical Sciences)

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