Fusion of FokI and catalytically inactive prokaryotic Argonautes enables site-specific programmable DNA cleavage.

Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, and robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif, but their lack of intrinsic dsDNA unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage, and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo.

Prebiotic N-(2-Aminoethyl)-Glycine (AEG)-Assisted Synthesis of Proto-RNA?

Quantum mechanical calculations are used to explore the thermodynamics of possible prebiotic synthesis of the building blocks of nucleic acids. Different combinations of D-ribofuranose (Ribf) and N-(2-aminoethyl)-glycine (AEG) (trifunctional connectors (TCs)); the nature of the Ribf, its anomeric form, and its ring puckering (conformation); and the nature of the nucleobases (recognition units (RUs)) are considered. The combinatorial explosion of possible nucleosides has been drastically reduced on physicochemical grounds followed by a detailed thermodynamic evaluation of alternative synthetic pathways. The synthesis of nucleosides containing N-(2-aminoethyl)-glycine (AEG) is predicted to be thermodynamically favored suggesting a possible role of AEG as a component of an ancestral proto-RNA that may have preceded today's nucleic acids. A new pathway for the building of free nucleotides (exemplified by 5'-uridine monophosphate (UMP)) and of AEG dipeptides is proposed. This new pathway leads to a spontaneous formation of free UMP assisted by an AEG nucleoside in an aqueous environment. This appears to be a workaround to the "water problem" that prohibits the synthesis of nucleotides in water.

Hybrid peptide-PNA monomers as building blocks for the fabrication of supramolecular aggregates.

Advantages like biocompatibility, biodegradability and tunability allowed the exploitation of peptides and peptidomimetics as versatile therapeutic or diagnostic agents. Because of their selectivity towards transmembrane receptors or cell membranes, peptides have also been identified as suitable molecules able to deliver in vivo macromolecules, proteins or nucleic acids. However, after the identification of the homodimer diphenylalanine (FF) as an aggregative motif inside the Aß1-42 polypeptide, short and ultrashort peptides have been studied as building blocks for the fabrication of supramolecular, ordered nanostructures for applications in biotechnological, biomedical and industrial fields. In this perspective, many hybrid molecules that combine FF with other chemical entities have been synthesized and characterized. Two novel hybrid derivatives (tFaF and cFgF), in which the FF homodimer is alternated with the peptide-nucleic acid (PNA) heterodimer "g-c" (guanine-cytosine) or "a-t" (adenine-thymine) and their dimeric forms (tFaF)2 and (cFgF)2 were synthesized. The structural characterization performed by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopies highlighted the capability of all the FF-PNA derivatives to self-assemble into ß-sheet structures. As a consequence of this supramolecular organization, the resulting aggregates also exhibit optoelectronic properties already reported for other similar nanostructures. This photoemissive behavior is promising for their potential applications in bioimaging.

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.

Two-Helix Supramolecular Proteomimetic Binders Assembled via PNA-Assisted Disulfide Crosslinking.

Peptidic motifs folded in a defined conformation are able to inhibit protein-protein interactions (PPIs) covering large interfaces and as such they are biomedical molecules of interest. Mimicry of such natural structures with synthetically tractable constructs often requires complex scaffolding and extensive optimization to preserve the fidelity of binding to the target. Here, we present a novel proteomimetic strategy based on a 2-helix binding motif that is brought together by hybridization of peptide nucleic acids (PNA) and stabilized by a rationally positioned intermolecular disulfide crosslink. Using a solid phase synthesis approach (SPPS), the building blocks are easily accessible and such supramolecular peptide-PNA helical hybrids could be further coiled using precise templated chemistry. The elaboration of the structural design afforded high affinity SARS CoV-2 RBD (receptor binding domain) binders without interference with the underlying peptide sequence, creating a basis for a new architecture of supramolecular proteomimetics.

Improved Triplex-Forming Isoorotamide PNA Nucleobases for A-U Recognition of RNA Duplexes.

Four new isoorotamide (Io)-containing PNA nucleobases have been designed for A-U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A-U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A-binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A-binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.

From Peptide Nucleic Acids to Supramolecular Structures of Nucleic Acid Derivatives.

Nucleic acids play a pivotal role in life processes. The endeavours to shed light on the essential properties of these intriguing building blocks led us to the synthesis of different analogues and the investigation of their properties. First various peptide nucleic acid monomers and oligomers have been synthesized, using an Fmoc/acyl protecting group strategy, and their properties studied. The serendipitous discovery of a side reaction of coupling agents led us to the elaboration of a peptide sequencing method. The capricious behaviour of guanine derivatives spurred the determination of their substitution pattern using 13 C, 15 N NMR, and mass spectrometric methods. The properties of guanines initiated the logical transition to the study of supramolecular systems composed of purine analogues. Thus, xanthine and uracil derivatives have been obtained and their supramolecular self-assembly properties scrutinized in gas, solid, and liquid states and at solid-liquid interfaces.

Peptide Nucleic Acids: Recent Developments in the Synthesis and Backbone Modifications.

Nucleic acid represents the ideal drug candidate for protein targets that are hard to target or against which drug development is not easy. Peptide nucleic acids (PNAs) are synthesized by attaching modified peptide backbones generally derived from repetitive N-2-aminoethyl glycine units in place of the regular phosphodiester backbone and represent synthetic impersonator of nucleic acids that offers an exciting research field due to their fascinating spectrum of biotechnological, diagnostic and potential therapeutic applications. The semi-rigid peptide nucleic acid backbone serves as a nearly-perfect template for attaching complimentary base pairs on DNA or RNA in a sequence-dependent manner as described by Watson-Crick models. PNAs and their analogues are endowed with exceptionally high affinity and specificity for receptor sites, essentially due to their polyamide backbone's uncharged and flexible nature. The present review compiled various strategies to modify the polypeptide backbone for improving the target selectivity and stability of the PNAs in the body. The investigated biological activities carried out on PNAs have also been summarized in the present review.

Head on Comparison of Self- and Nano-assemblies of Gamma Peptide Nucleic Acid Amphiphiles.

Peptide nucleic acids (PNAs) are nucleic acid analogs with superior hybridization properties and enzymatic stability than deoxyribonucleic acid (DNA). In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymer due to the Watson-Crick -based molecular recognition and flexibility of synthesis. Here, we engineered PNA amphiphiles using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γPNA) amphiphiles self-assemble into spherical vesicles. Further, we formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols. We perform comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and nano-assemblies. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) analysis reveal ellipsoidal morphology of γPNA nano-assemblies that results in superior cellular delivery compate to the spherical self-assembly. Next, we compare the functional activities of γPNA self-and nano-assemblies in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays. Overall, we establish that γPNA amphiphile is a functionally active bio-polymer to formulate nano-assemblies for a wide range of biomedical applications.

Development of Surface Chemical Strategies for Synthesizing Redox-Responsive Diatomite Nanoparticles as a Green Platform for On-Demand Intracellular Release of an Antisense Peptide Nucleic Acid Anticancer Agent.

Redox-responsive silica drug delivery systems are synthesized by aeco-friendly diatomite source to achieve on-demand release of peptide nucleic acid (PNA) in tumor reducing microenvironment, aiming to inhibit the immune checkpoint programmed cell death 1 receptor/programmed cell death receptor ligand 1 (PD-1/PD-L1) in cancer cells. The nanoparticles (NPs) are coated with polyethylene glycol chains as gatekeepers to improve their physicochemical properties and control drug release through the cleavable disulfide bonds (S-S) in a reductive environment. This study describes different chemical conditions to achieve the highest NPs' surface functionalization yield, exploring both multistep and one-pot chemical functionalization strategies. The best formulation is used for covalent PNA conjugation via the S-S bond reaching a loading degree of 306 ± 25 µg PNA mg-1 DNPs . These systems are used for in vitro studies to evaluate the kinetic release, biocompatibility, cellular uptake, and activity on different cancer cells expressing high levels of PD-L1. The obtained results prove the safety of the NPs up to 200 µg mL-1 and their advantage for controlling and enhancing the PNA intracellular release as well as antitumor activity. Moreover, the downregulation of PD-L1 observed only with MDA-MB-231 cancer cells paves the way for targeted immunotherapy.

Fluorobenzene Nucleobase Analogues for Triplex-Forming Peptide Nucleic Acids.

2,4-Difluorotoluene is a nonpolar isostere of thymidine that has been used as a powerful mechanistic probe to study the role of hydrogen bonding in nucleic acid recognition and interactions with polymerases. In the present study, we evaluated five fluorinated benzenes as nucleobase analogues in peptide nucleic acids designed for triple helical recognition of double helical RNA. We found that analogues having para and ortho fluorine substitution patterns (as in 2,4-difluorotoluene) selectively stabilized Hoogsteen triplets with U-A base pairs. The results were consistent with attractive electrostatic interactions akin to non-canonical F to H-N and C-H to N hydrogen bonding. The fluorinated nucleobases were not able to stabilize Hoogsteen-like triplets with pyrimidines in either G-C or A-U base pairs. Our results illustrate the ability of fluorine to engage in non-canonical base pairing and provide insights into triple helical recognition of RNA.

Combined Treatment of Bronchial Epithelial Calu-3 Cells with Peptide Nucleic Acids Targeting miR-145-5p and miR-101-3p: Synergistic Enhancement of the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a "combo-therapy" associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.

Extended Peptide Nucleic Acid Nucleobases Based on Isoorotic Acid for the Recognition of A-U Base Pairs in Double-Stranded RNA.

Peptide nucleic acids (PNA) with extended isoorotamide containing nucleobases (Io ) were designed for binding A-U base pairs in double-stranded RNA. Isothermal titration calorimetry and UV thermal melting experiments revealed improved affinity for A-U using the Io scaffold in PNA. PNAs having four sequential Io extended nucleobases maintained high binding affinity.

Peptide nucleic acids and their role in gene regulation and editing.

The unique properties of peptide nucleic acid (PNA) makes it a desirable candidate to be used in therapeutic and biotechnological interventions. It has been broadly utilized for numerous applications, with a major focus in regulation of gene expression, and more recently in gene editing. While the classic PNA design has mainly been employed to date, chemical modifications of the PNA backbone and nucleobases provide an avenue to advance the technology further. This review aims to discuss the recent developments in PNA based gene manipulation techniques and the use of novel chemical modifications to improve the current state of PNA mediated gene targeting.

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.

Multivalent dextran hybrids for efficient cytosolic delivery of biomolecular cargoes.

The development of novel biotherapeutics based on peptides and proteins is often limited to extracellular targets, because these molecules are not able to reach the cytosol. In recent years, several approaches were proposed to overcome this limitation. A plethora of cell-penetrating peptides (CPPs) was developed for cytoplasmic delivery of cell-impermeable cargo molecules. For many CPPs, multimerization or multicopy arrangement on a scaffold resulted in improved delivery but also higher cytotoxicity. Recently, we introduced dextran as multivalent, hydrophilic polysaccharide scaffold for multimerization of cell-targeting cargoes. Here, we investigated covalent conjugation of a CPP to dextran in multiple copies and assessed the ability of resulted molecular hybrid to enter the cytoplasm of mammalian cells without largely compromising cell viability. As a CPP, we used a novel, low-toxic cationic amphiphilic peptide L17E derived from M-lycotoxin. Here, we show that cell-penetrating properties of L17E are retained upon multivalent covalent linkage to dextran. Dextran-L17E efficiently mediated cytoplasmic translocation of an attached functional peptide and a peptide nucleic acid (PNA). Moreover, a synthetic route was established to mask the lysine side chains of L17E with a photolabile protecting group thus opening avenues for light-triggered activation of cellular uptake.

Peptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular Imaging.

Protease-triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnology. Herein, we report the design of a protease-activatable aptamer system that can perform molecular sensing and imaging in a tumor-specific manner. The system is constructed by locking the structure-switching activity of an aptamer using a rationally designed PNA-peptide-PNA triblock copolymer. Highly selective protease-mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell-selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.

Efficient Gene Therapy of Pancreatic Cancer via a Peptide Nucleic Acid (PNA)-Loaded Layered Double Hydroxides (LDH) Nanoplatform.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignant tumors with extremely poor prognosis due to the later stage diagnosis when surgical resection is no longer applicable. Alternatively, the traditional gene therapy which drives pancreatic cancer cells into an inactive state and inhibiting the proliferation and metastasis, presents potentials to safely inhibit pancreatic cancer progression, but unfortunately has received limited success to date. Here, an efficient gene therapy of pancreatic cancer is shown via a peptide nucleic acid (PNA)-loaded layered double hydroxides (LDHs) nanoplatform. Compared with the traditional DNA- or RNA-based gene therapies, the gene therapy using PNA features great advantages in recognizing and hybridizing with the target mutant sequences to form PNA-DNA hybrids with significantly enhanced stability due to the absence of electrostatic repulsion, and the constrained flexibility of the polyamide backbone. Moreover, ultrasmall LDHs are engineered to load PNA and the obtained PNA-loaded LDH platform (LDHs/PNA) is capable of efficiently and selectively targeting the intranuclear mutant sequences thanks to the proton sponge effect. Treatments with LDHs/PNA demonstrate markedly inhibited growth of pancreatic cancer xenografts via a cancer cell proliferation suppression mechanism. The results demonstrate the great potentials of LDHs/PNA as a highly promising gene therapy agent for PDAC.