Furan-PNA : a mildly inducible irreversible interstrand crosslinking system targeting single and double stranded DNA

We here report on the design and synthesis of tailor-made furan-modified peptide nucleic acid (PNA) probes for covalent targeting of single stranded DNA through a crosslinking strategy. After introducing furan-containing building blocks into a PNA sequence, hybridization and furan-oxidation based crosslinking to DNA is investigated. The structure of the crosslinked products is characterized and preliminary investigations concerning the application of these systems to double stranded DNA are shown

Exploiting double exchange Diels-Alder cycloadditions for immobilization of peptide nucleic acids on gold nanoparticles

The generation of PNA-decorated gold nanoparticles (AuNPs) has revealed to be more difficult as compared to the generation of DNA-functionalized ones. The less polar nature of this artificial nucleic acid system and the associated tendency of the neutral poly-amidic backbone to aspecifically adsorb onto the gold surface rather than forming a covalent bond through gold-thiol interaction, combined with the low solubility of PNAs itself, form the main limiting factors in the functionalization of AuNP. Here, we provide a convenient methodology that allows to easily conjugate PNAs to AuNP. Positively charged PNAs containing a masked furan moiety were immobilized via a double exchange Diels-Alder cycloaddition onto masked maleimide-functionalized AuNPs in a one-pot fashion. Conjugated PNA strands retain their ability to selectively hybridize with target DNA strands. Moreover, the duplexes resulting from hybridization can be detached through a retro-Diels-Alder reaction, thus allowing straightforward catch-and-release of specific nucleic acid targets

PNA-based microRNA detection methodologies

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

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

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

Effect of chirality in gamma-PNA: PNA interaction, another piece in the picture

Modification of the PNA backbone can be used to broaden their utility by introducing new functional groups. In particular, gamma-modified PNA have been found to be quite effective in a number of applications, and exhibit particularly high DNA binding affinity. The introduction of one side chain imply that the achiral backbone of PNA becomes chiral, and binding properties depend on the stereochemistry. A new paper on gamma-modified PNA by Ly and co-workers complete the existing knowledge by displaying that in binding to complementary PNA stereochemical orthogonality can be demonstrated. This opens the way to the exploitation of stereochemical features in diagnostic assays and in nanofabrication

Hydrolysis of 5-methylfuran-2-yl to 2,5-dioxopentanyl allows for stable bio-orthogonal proximity-induced ligation

Proximity-based ligations commonly require an external stimulus such as a catalyst or irradiation, or highly reactive functional groups. Here the reaction of alpha effect nucleophiles and 2,5-dioxopentanyl derivatives allows direct proximity-based ligation while avoiding highly reactive moieties. Ligation methodologies featuring bio-orthogonal units and leading to the formation of a stable adduct are the ideal candidates for being applied in a biological context. However, most of the available strategies rely on highly reactive species that require careful handling, or on the activation of pro-reactive functional groups. We here report on a proximity-induced ligation reaction that relies on a stable 2,5-dione, that can be conveniently generated under acidic conditions from a 2,5-dialkylfuran building block, and hydrazine nucleophiles. This bio-orthogonal ligation, which proceeds under physiological conditions, does not require any stimulus or trigger and leads to the formation of a pyridazinium adduct that demonstrates excellent stability under harsh conditions (24 h at 90 degrees C). The reaction was applied to the formation of PNA-PNA adducts, DNA- and RNA-templated ligations, and for the formation of peptide-peptide adducts in solution. This convenient methodology was further implemented on plastic and glass surfaces to realize self-addressable covalent constructs

Visible-light triggered templated ligation on surface using furan-modified PNAs

Oligonucleotide-templated reactions are frequently exploited for target detection in biosensors and for the construction of DNA-based materials and probes in nanotechnology. However, the translation of the specifically used template chemistry from solution to surfaces, with the final aim of achieving highly selective high-throughput systems, has been difficult to reach and therefore, poorly explored. Here, we show the first example of a visible light-triggered templated ligation on a surface, employing furan-modified peptide nucleic acids (PNAs). Tailored photo-oxidation of the pro-reactive furan moiety is ensured by the simultaneous introduction of a weak photosensitizer as well as a nucleophilic moiety in the reacting PNA strand. This allows one to ensure a localized production of singlet oxygen for furan activation, which is not affected by probe dilution or reducing conditions. Simple white light irradiation in combination with target-induced proximity between reactive functionalities upon recognition of a short 22mer DNA or RNA sequence that functions as a template, allows sensitive detection of nucleic acid targets in a 96 well plate format

Efficient cell penetration and delivery of peptide nucleic acids by an argininocalix[4]arene

The application of Peptide Nucleic Acids (PNAs), mimics of DNA lacking the sugar-phosphate backbone, for antisense/anti-gene therapy and gene editing is limited by their low uptake by cells. Currently, no simple and efficient delivery systems and methods are available to solve this open issue. One of the most promising approach is the modification of the PNA structure through the covalent linkage of poliarginine tails, but this means that every PNA intended to be internalized must be modified. Herein we report the results relative to the delivery ability of a macrocyclic multivalent tetraargininocalix[4]arene (1) used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity and ease preparation of the transfection formulation, simply attained by mixing PNA and calixarene, candidate this vector as universal delivery system for this class of nucleic acid analogues

Pyrene-modified PNAs: Stacking interactions and selective excimer emission in PNA2DNA triplexes

Pyrene derivatives can be incorporated into nucleic acid analogs in order to obtain switchable probes or supramolecular architectures. In this paper, peptide nucleic acids (PNAs) containing 1 to 3 1-pyreneacetic acid units (PNA1-6) with a sequence with prevalence of pyrimidine bases, complementary to cystic fibrosis W1282X point mutation were synthesized. These compounds showed sequence-selective switch-on of pyrene excimer emission in the presence of target DNA, due to PNA(2)DNA triplex formation, with stability depending on the number and positioning of the pyrene units along the chain. An increase in triplex stability and a very high mismatch-selectivity, derived from combined stacking and base-pairing interactions, were found for PNA2, bearing two distant pyrene units