Synthesis and stability studies of bicyclo[6.1.0]nonyne scaffolds for automated solid-phase oligonucleotide synthesis.

Two novel bicyclo[6.1.0]nonyne (BCN) linker derivatives, which can be directly incorporated into oligonucleotide sequences during standard automated solid-phase synthesis, are reported.

Zinc ,-bis(2-picolyl)amine Chelates Show Substitution-Dependent Cleavage of Phosphodiesters in Models as Well as of PNAzyme-RNA Bulges.

PNAzymes are a group of artificial enzymes which show promising results in selective and efficient cleavage of RNA targets. In the present study, we introduce a series of metal chelating groups based on ,-bis(2-picolyl) groups (parent, 6-methyl and 6-amino substituted) as the active sites of novel PNAzymes. An improved synthetic route for the 6-amino analogues is described.

G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform.

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties.

Influence of sequence variation on the RNA cleavage activity of Zn-dimethyl-dppz-PNA-based artificial enzymes.

The development of Zn-dependent dimethyl-dppz-PNA conjugates (PNAzymes) as efficient site-specific artificial ribonucleases enables rapid sequence-specific degradation of clinically relevant RNA target sequences, but the significance of the RNA/PNAzyme sequence and structural demands for the identification of novel RNA targets are not fully understood. In the present study, we investigated the influence of sequence variation in the recognition arms of the RNA/PNAzyme complex on the RNA cleavage activity of the artificial enzymes. The base pairs closing the 3-nucleotide bulge region on both sides of the bulge as well as the neighbouring nucleobases were shown to significantly influence the RNA cleavage activity.

2'--(-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity.

2'--(-(Aminoethyl)carbamoyl)methyl (2'--AECM)-modified oligonucleotides (ONs) and their mixmers with 2'--methyl oligonucleotides (2'-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages.

NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution.

The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted endocytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA.

Innovative developments and emerging technologies in RNA therapeutics.

RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020.

A Study on Synthesis and Upscaling of 2'--AECM-5-methyl Pyrimidine Phosphoramidites for Oligonucleotide Synthesis.

2'--(-(Aminoethyl)carbamoyl)methyl-modified 5-methyluridine (AECM-MeU) and 5-methylcytidine (AECM-MeC) phosphoramidites are reported for the first time and prepared in multigram quantities. The syntheses of AECM-MeU and AECM-MeC nucleosides are designed for larger scales (approx. 20 g up until phosphoramidite preparation steps) using low-cost reagents and minimizing chromatographic purifications.

Zn-Dependent peptide nucleic acid-based artificial ribonucleases with unprecedented efficiency and specificity.

We present Zn-dependent dimethyl-dipyridophenazine PNA conjugates as efficient RNA cleaving artificial enzymes. These PNAzymes display site-specific RNA cleavage with 10 minute half-lives and cleave clinically relevant RNA models.

S-SIL of PCSK9-Active Oligonucleotide as Tools for Accurate Quantification by Mass Spectrometry.

Stable isotope labeling (SIL) of active pharmaceutical ingredients (API) is a well-established technique for the accurate quantification of small-molecule drugs. As the scope of active ingredients is expanding into areas of larger molecules, such as oligonucleotides (ONs), the development of new quantification techniques is critical. Herein, we describe the analysis of a S-SIL anti-PCSK9 gapmer-type antisense ON.

The Mechanism of Cleavage of RNA Phosphodiesters by a Gold Nanoparticle Nanozyme.

The cleavage of uridine 3'-phosphodiesters bearing alcohols with pK ranging from 7.14 to 14.5 catalyzed by AuNPs functionalized with 1,4,7-triazacyclononane-Zn(II) complexes has been studied to unravel the source of catalysis by these nanosystems (nanozymes).

New Alkyne and Amine Linkers for Versatile Multiple Conjugation of Oligonucleotides.

Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly.

Synthesis and biological evaluation of modified laminin peptide (NS-KDP) with enhanced affinity for neuronal growth and targeted molecular imaging (SPECT).

An analog of γ1 laminin (RDIAEIIKDI) decapeptide has been used to augment neuronal survival and regeneration after injuries, during aging and other CNS disorder. As a prime synthetic peptide, KDI, is responsible for the neurite outgrowth of human embryonic neurons. In this study, we have designed, modified a KDI derivative and synthesized by replacing isoleucine (I) with Pro (P) amino acid at C-terminal to enhance its potency towards neurite growth.

Copper-Catalyzed Huisgen 1,3-Dipolar Cycloaddition Tailored for Phosphorothioate Oligonucleotides.

An efficient method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition ("click reaction"). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this conjugation method has to be adjusted to be compatible with the sulfur-containing groups. The method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me S) as a mediator for the click reaction.

Attachment of Peptides to Oligonucleotides on Solid Support Using Copper(I)-Catalyzed Huisgen 1,3-Dipolar Cycloaddition.

In vivo bioavailability and delivery of nucleic acids to the site of action is a severe limitation in oligonucleotide (ON) therapeutics. Equipping the ONs with cell penetrating, homing or endosomal escape peptides can enhance specificity and/or uptake efficiencies. We describe here a general procedure for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support utilizing a novel activated alkyne containing linker which enhances the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition.

Novel aroylated phenylenediamine compounds enhance antimicrobial defense and maintain airway epithelial barrier integrity.

Aroylated phenylenediamines (APDs) are novel inducers of innate immunity enhancing cathelicidin gene expression in human bronchial epithelial cell lines. Here we present two newly developed APDs and aimed at defining the response and signaling pathways for these compounds with reference to innate immunity and antimicrobial peptide (AMP) expression. Induction was initially defined with respect to dose and time and compared with the APD Entinostat (MS-275).

Efficient Conjugation to Phosphorothioate Oligonucleotides by Cu-Catalyzed Huisgen 1,3-Dipolar Cycloaddition.

Improving oligonucleotide delivery is critical for the further development of oligonucleotide-based therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches toward efficient oligonucleotide-based therapies. An efficient methods for the attachment of a variety of reporter groups is Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition.

Further Probing of Cu-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes.

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems.

Amyloid-β Peptide Targeting Peptidomimetics for Prevention of Neurotoxicity.

A new generation of ligands designed to interact with the α-helix/β-strand discordant region of the amyloid-β peptide (Aβ) and to counteract its oligomerization is presented. These ligands are designed to interact with and stabilize the Aβ central helix (residues 13-26) in an α-helical conformation with increased interaction by combining properties of several first-generation ligands. The new peptide-like ligands aim at extended hydrophobic and polar contacts across the central part of the Aβ, that is, "clamping" the target.

A Versatile and Convenient Synthesis of S-Labeled Phosphorothioate Oligonucleotides.

A synthetic protocol for S-labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS-based assay analysis. This was enabled by a highly efficient, two-step, one-pot synthesis of S-labeled phenylacetyl disulfide ( S-PADS), starting from S-enriched elemental sulfur ( S ). S-PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone.

Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA-Bulge Size and Sequence Dependence.

In this report, we investigate the efficiency and selectivity of a Zn-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge.

Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection.

Treatment with Entinostat Heals Experimental Cholera by Affecting Physical and Chemical Barrier Functions of Intestinal Epithelia.

We have shown previously that oral treatment with sodium butyrate or phenylbutyrate in an experimental model of shigellosis improves clinical outcomes and induces the expression of the antimicrobial peptide CAP-18 in the large intestinal epithelia. In a subsequent study, we found that entinostat, an aroylated phenylenediamine compound, has similar therapeutic potential against shigellosis. In this study, we aimed to evaluate entinostat as a potential candidate for host-directed therapy against cholera in an experimental model.

F NMR Spectroscopic Analysis of the Binding Modes in Triple-Helical Peptide Nucleic Acid (PNA)/MicroRNA Complexes.

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA) /RNA binding modes could be obtained.