Applications Of Peptide Nucleic Acids Research Articles

Peptide Nucleic Acids: From Origami to Editing.

Peptide nucleic acids (PNAs) combine the programmability of native nucleic acids with the robustness and ease of synthesis of a peptide backbone. These designer biomolecules have demonstrated tremendous utility across a broad range of applications, from the formation of bespoke biosupramolecular architectures to biosensing and gene regulation. Herein, we explore some of the key developments in the application of PNA in chemical biology and biotechnology in the last 5 years and present anticipated key areas of future development.

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

Recent progress in PNA-based biosensing technology

<p indent="0mm">Peptide nucleic acid (PNA) is a synthetic oligonucleotide analog composed of nucleobases arrayed along a pseudopeptide backbone. It can integrate the power of nucleic acids to encode information with the versatility of amino acids to encode structure and function. PNA has high affinity and specificity for hybridizing with complementary nucleic acid sequences, leading to stable PNA-DNA or PNA-RNA duplex structures. PNA is expected to overcome the limitations of nucleic acids including low stability and specificity in biomedical applications. In this review, we firstly give a brief introduction to the unique properties of PNA and its biomedical applications. Next, we describe and highlight the recent progress in biosensing applications of PNA and DNA integration. Lastly, the future prospects and challenges are discussed.

Mechanisms and applications of peptide nucleic acids selectively binding to double-stranded RNA.

RNAs form secondary structures containing double-stranded base paired regions and single-stranded regions. Probing, detecting and modulating RNA structures and dynamics requires the development of molecular sensors that can differentiate the sequence and structure of RNAs present in viruses and cells, as well as in extracellular space. In this review, we summarize the recent progress on the development of chemically modified peptide nucleic acids (PNAs) for the selective recognition of double-stranded RNA (dsRNA) sequences over both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) sequences. We also briefly discuss the applications of sequence-specific dsRNA-binding PNAs in sensing and stabilizing dsRNA structures and inhibiting dsRNA-protein interactions.

Peptide nucleic acid as a template for Taq DNA polymerase

Peptide nucleic acid (PNA), an artificial DNA analog, comprises a purine or pyrimidine base and a pseudo-peptide backbone instead of deoxyribose-phosphate. PNA has been found to have stronger adhesion and higher stability in binding to its complementary DNA than deoxyribose-phosphate. Thus, it could serve as an agent for gene modulation, demonstrating potential in antisense therapy, molecular diagnostics, and nanotechnology. However, the applications of PNA remain limited because its biological activities are not fully known. Here, I demonstrate that a thermostable DNA polymerase, Thermus aquaticus (Taq) polymerase, exhibits transcriptase activity when a PNA oligomer is used as a template and that genetic information of the oligomer can be amplified by PCR using DNA primers. Furthermore, the insertion of a glutamine peptide stretch in the middle part of the PNA template did not interfere with transcription; it was transcribed into a guanosine or adenosine stretch. Intriguingly, this amino acid-to-DNA transcription did not occur when glycine residues were inserted. A synthetic PNA oligomer can, therefore, function as a template for a DNA polymerase, and polyglutamine peptides can be transcribed into guanosine or adenosine. These findings provide a cornerstone to reveal all amino acid genetic codes and transcription activity in the future.

Novel peptide (RATH) mediated delivery of peptide nucleic acids for antiviral interventions.

The peptide nucleic acid (PNA) is a chimeric molecule with the nucleobases connected by peptide bonds. This chimeric nature gives the PNA certain therapeutic advantages over natural antisense nucleic acid molecules. The PNA probes are known for its better and stronger complementation with target nucleic acids. However, cellular delivery of PNA is a major hurdle due to the charge-neutral nature of the PNA. For cellular delivery of PNA, peptide-PNA conjugates are used. This approach may face some practical limitation in terms of PNA antisense activity. In this study, we propose a novel RATH-2 peptide-based non-covalent PNA delivery mechanism. We observed RATH-2 shows a favorable molecular interaction with PNA at 16:1 (peptide:PNA) molar ratio resulting in co-centric nanoparticle formation. With this combination, we could achieve as high as 93% cellular delivery of the PNA. The proposed non-covalent RATH:PNA delivery model showed endocytic entrapment free delivery of PNA. The study further demonstrated the therapeutic application of PNA with in vitro antiviral intervention model. Using RATH-2 non-covalent PNA delivery system, we could inhibit 69.5% viral load. The present study demonstrates a cell-penetrating peptide:PNA interaction can lead to nanoparticle formations that facilitated cellular delivery of PNA.Key points• A novel cell-penetrating peptide (RATH-2) was identified for non-covalent delivery of PNA.• RATH-2 and PNA formed co-centric nanoparticles at appropriate molar combination.• PNA delivered through the RATH-2 inhibited the viral gene expression and reduced the viral load.

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications.

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.

Application of peptide nucleic acid in electrochemical nucleic acid biosensors.

The early diagnosis of major diseases, such as malignant tumors, has always been an important field of research. Through screening, early detection of such diseases, and timely and effective treatment can significantly improve the survival rate of patients and reduce medical costs. Therefore, the development of a simple detection method with high sensitivity and strong specificity, and that is low cost is of great significance for the diagnosis and prognosis of the disease. Electrochemical DNA biosensing analysis is a technology based on Watson Crick base complementary pairing, which uses the capture probe of a known sequence to specifically recognize the target DNA and detect its concentration. Because of its advantages of low cost, simple operation, portability, and easy miniaturization, it has been widely researched and has become a cutting-edge topic in the field of biochemical analysis and precision medicine. However, the existing methods for electrochemical DNA biosensing analysis have some shortcomings, such as poor stability and specificity of capture probes, insufficient detection sensitivity, and long detection cycles. In this review, we focus on improving the sensitivity and practicability of electrochemical DNA biosensing analysis methods and summarize a series of research work carried out by using electrically neutral peptide nucleic acid as an immobilized capture probe.

Rapid self-assembly of γPNA nanofibers at constant temperature.

Peptide nucleic acids (PNAs) have primarily been used to achieve therapeutic gene modulation through antisense strategies since their design in the 1990s. However, the application of PNAs as a functional nanomaterial has been more recent. We recently reported that γ-modified peptide nucleic acids (γPNAs) could be used to enable formation of complex, self-assembling nanofibers in select polar aprotic organic solvent mixtures. Here we demonstrate that distinct γPNA strands, each with a high density of γ-modifications can form complex nanostructures at constant temperatures within 30 minutes. Additionally, we demonstrate DNA-assisted isothermal growth of γPNA nanofibers, thereby overcoming a key hurdle for future scale-up of applications related to nanofiber growth and micropatterning.

Optimized Synthesis of Fmoc/Boc‐Protected PNA Monomers and their Assembly into PNA Oligomers.

AbstractContinuous advancement of application of peptide nucleic acid (PNA) oligomers encouraged exploration of rapid and efficient synthesis of PNA monomers and oligomers. Among the PNA monomers developed, only a few are commonly used in automated PNA synthesis. Herein, we report short and efficient protocols suitable for large‐scale synthesis of Fmoc/Boc‐protected PNA monomers with advantageous solubility properties; these also facilitate purification due to the traceless nature of the Boc protecting group. Initially, several coupling reagents were screened for assembly of a pentamer containing all four nucleobases, and then the most promising reagents were tested in the synthesis of a decamer. The Fmoc/Boc‐protected monomers proved compatible with both manual synthesis and assembly on an automated peptide synthesizer at room temperature or at 40 °C. As compared to the commonly used coupling agent, 1‐[bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxide hexafluorophosphate (HATU), both 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU) and [ethyl cyano(hydroxyimino)acetato−O2]tri‐1‐pyrrolidinylphosphonium hexafluorophosphate (PyOxim) proved more favorable, with the latter being superior. A previously reported side reaction of guanine bases in the presence of benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was not observed with the phosphonium salts.

Recognition of Target Site in Various Forms of DNA and RNA by Peptide Nucleic Acid (PNA): From Fundamentals to Practical Applications

Abstract Peptide nucleic acid (PNA) is a DNA analog, in which the sugar-phosphate backbone in DNA is replaced by poly[N-(2-aminoethyl)glycine]. Since its discovery in the early 1990s, PNA has been widely employed in chemistry, biochemistry, medicine, nanotechnology, and many other fields. This account surveys recent developments on the design of PNA derivatives and their applications. In the first part, PNAs for sequence-specific recognition of DNA and RNA (single-strands, double-strands, G-quadruplexes, i-motifs, and others) are comprehensively covered. Modifications of nucleobases and of the main chain effectively promote both the strength of binding and the selectivity of recognition. In the second half of this account, practical applications of PNA are presented. Structural restraints, induced by complex formation of PNA with DNA and RNA substrates, lead to selective transformation of target sites to desired structures. Applications to regulation of gene expression, gene editing, construction of sophisticated nanostructures, and others are also described. Advantages and disadvantages of PNAs, compared with other sequence-recognizing molecules hitherto reported, are discussed in terms of various physicochemical and biological features.

Multifunctional Delivery Systems for Peptide Nucleic Acids.

The number of applications of peptide nucleic acids (PNAs)—oligonucleotide analogs with a polyamide backbone—is continuously increasing in both in vitro and cellular systems and, parallel to this, delivery systems able to bring PNAs to their targets have been developed. This review is intended to give to the readers an overview on the available carriers for these oligonucleotide mimics, with a particular emphasis on newly developed multi-component- and multifunctional vehicles which boosted PNA research in recent years. The following approaches will be discussed: (a) conjugation with carrier molecules and peptides; (b) liposome formulations; (c) polymer nanoparticles; (d) inorganic porous nanoparticles; (e) carbon based nanocarriers; and (f) self-assembled and supramolecular systems. New therapeutic strategies enabled by the combination of PNA and proper delivery systems are discussed.

Walking through the wonder years of artificial DNA: peptide nucleic acid

Peptide Nucleic Acid (PNA) serves as an artificial functional analog of DNA. Being immune to enzymatic degradation and possessing strong affinity towards DNA and RNA, it is an ideal candidate for many medical and biotechnological applications that are of antisense and antigene in nature. PNAs are anticipated to have its application in DNA and RNA detection as well as quantification, to serve as antibacterial and antiviral agents, and silencing gene for developing anticancer strategies. Although, their restricted entry in both eukaryotic and prokaryotic cells limit their applications. In addition, aggregation of PNA in storage containers reduces the quality and quantity of functional PNA that makes it inadequate for their mass production and storage. To overcome these limitations, researchers have modified PNA either by the addition of diverse functional groups at various loci on its backbone, or by synthesizing chimeras with other moieties associated with various delivery agents that aids their entry into the cell. Here, this review article summarizes few of the structural modifications that are performed with PNA, methods used to improve their cellular uptake and shedding light on the applications of PNA in various prospects in biological sciences.

Development of Cu2+-Based Distance Methods and Force Field Parameters for the Determination of PNA Conformations and Dynamics by EPR and MD Simulations.

Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu2+ complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV-vis and circular dichroism measurements and assess the efficacy of the Cu2+ label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu2+ label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu2+ EPR at X-band. These results present for the first time a rigid Cu2+ spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large.

Potential applications of peptide nucleic acid in biomedical domain.

Peptide Nucleic Acid (PNA) are DNA/RNA synthetic analogs with 2‐([2‐aminoethyl] amino) acetic acid backbone. They partake unique antisense and antigene properties, just due to its inhibitory effect on transcription and translation; they also undergo complementary binding to RNA/DNA with high affinity and specificity. Hence, to date, many methods utilizing PNA for diagnosis and treatment of various diseases namely cancer, AIDS, human papillomavirus, and so on, have been designed and developed. They are being used widely in polymerase chain reaction modulation/mutation, fluorescent in‐situ hybridization, and in microarray as a probe; they are also utilized in many in‐vitro and in‐vivo assays and for developing micro and nano‐sized biosensor/chip/array technologies. Earlier reviews, focused only on PNA properties, structure, and modifications related to diagnostics and therapeutics; our review emphasizes on PNA properties and synthesis along with its potential applications in diagnosis and therapeutics. Furthermore, prospects in biomedical applications of PNAs are being discussed in depth.

Application of Peptide Nucleic Acid in Virus Detection and Therapy

Application of Peptide Nucleic Acid in Virus Detection and Therapy

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

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.

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

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.

Poly(Lactic-co-Glycolic Acid) Nanoparticle Delivery of Peptide Nucleic Acids In Vivo.

Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting-the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations-as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs.

Advances in Nanoparticle-based Delivery of Next Generation Peptide Nucleic Acids.

Peptide nucleic acids (PNAs) belong to the next generation of synthetic nucleic acid analogues. Their high binding affinity and specificity towards the target DNA or RNA make them the reagent of choice for gene therapy-based applications. To review important gene therapy based applications of regular and chemically modified peptide nucleic acids in combination with nanotechnology. Selective research of the literature. Poor intracellular delivery of PNAs has been a significant challenge. Among several delivery strategies explored till date, nanotechnology-based strategies hold immense potential. Recent studies have shown that advances in nanotechnology can be used to broaden the range of therapeutic applications of PNAs. In this review, we discussed significant advances made in nanoparticle-based on PLGA polymer, silicon, oxidized carbon and graphene oxide for the delivery of PNAs. Nanoparticles delivered PNAs can be implied in diverse gene therapy based applications including gene editing as well as gene targeting (antisense) based strategies.