C5-Propynyl modified 2'-fluoroarabinonucleic acids form stable duplexes with RNA that are RNase H competent.

The clinical success of the antisense approach for the treatment of genetic disorders is indisputably the result of chemical modifications along the oligonucleotide (ON) scaffold, which impart desirable properties including high RNA affinity, nuclease stability and improved drug delivery. While effective, many modifications are not capable of eliciting an RNase H response limiting their application in antisense systems. To contribute to the structural design and inventory of nucleoside analogues with favorable antisense properties, herein we describe the synthesis of C5-propynyl-2'-fluoroarabinonucleic acids (FANAP). Incorporation of individual and multiple uridine (FaraUP) and cytidine (FaraCP) inserts into ONs revealed, both stabilized duplexes formed with RNA. In contrast, these modifications demonstrated a negligible (FaraUP) or reduced (FaraCP) effect on DNA binding. Moreover, modified ONs containing these analogues supported E. coli RNase H cleavage of RNA with an altered cleavage pattern observed relative to controls. Moreover, a 2'-O-methoxyethyl (2'-O-MOE) gapmer with a FANAP core was able to elicit RNA cleavage at an increased rate compared to C5-propynyl-arabinonucleic acids (ANAP). Enzymatic hydrolysis of these gapmers was assessed with nuclease S1 digestion and revealed greater stability of ANAP compared to FANAP. These results suggest C5-propynyl ANA/FANA modifications demonstrate promising potential for the design of therapeutic ONs.

Oligonucleotides Containing C5-Propynyl Modified Arabinonucleic Acids: Synthesis, Biophysical and Antisense Properties.

The introduction of chemical modifications on the nucleic acid scaffold has allowed for the progress of antisense oligonucleotides (ASOs) in the clinic for the treatment of a variety of disorders. In contribution to the repertoire of gene-silencing nucleic acid modifications, herein we report the synthesis and incorporation of C5-propynyl arabinouridine (araUP ) and arabinocytidine (araCP ) into mixed-base ASOs containing a pyrimidine core. Substitution of the core with araUP and araCP resulted in stabilization of the duplex formed with RNA but not with DNA. Similar results were obtained with ASOs bearing phosphorothioate linkages or methoxyethyl (MOE) wings in a gapmer design. All modified ASOs were compatible with E. coli RNase H mediated degradation of target RNA. Substitution of DNA for araUP and araCP in the central portion of a gapmer with MOE wings demonstrated improved nuclease resistance. These results suggest C5-modified arabinonucleic acids may serve as a potential chemical modification for therapeutic ASOs.

Preparation of a Convertible Spacer Containing a Disulfide Group for Versatile Functionalization of Oligonucleotides.

The protocols described in this article provide details regarding the synthesis and characterization of a disulfide containing linker phosphoramidite for terminal functionalization of synthetic oligonucleotides. The linker is first synthesized from 6-mercaptohexanol in two steps and is incorporated at the 5' end of short DNA oligonucleotides using automated solid-phase synthesis. The linker contains a terminal tosylate group which is post-synthetically displaced by altering the deprotection conditions to yield a variety of functional handles (N3 , NH2 , OMe, SH) or alternatively, the tosylate can be displaced directly with primary amines such as tert-butylamine. The linker system is also compatible with RNA oligonucleotides enabling the introduction of various functional handles (N3 , NH2 ). The protocol outlined in this procedure provides access to a versatile linker for the terminal functionalization of oligonucleotides containing a disulfide bond which may serve useful in the synthesis of reduction-responsive oligonucleotide conjugates. As a proof of concept, in this protocol the linker is used to modify a dT10 oligonucleotide and then conjugated by copper(I)-mediated azide-alkyne cycloaddition (CuAAC) to an alkyne-modified poly(ethylene glycol) which shows concentration dependent release of the oligonucleotide upon treatment with 1,4-dithiothreitol, a reducing agent. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of disulfide linker phosphoramidite 3 Basic Protocol 2: Synthesis, functionalization, and characterization of DNA oligonucleotides containing disulfide linker phosphoramidite 3 Basic Protocol 3: Displacement of terminal tosylate functionalized DNA with primary aliphatic amines Basic Protocol 4: Synthesis of oligonucleotide-PEG conjugate Support Protocol: Preparation of PEG-alkyne.

Synthesis of a Convertible Linker Containing a Disulfide Group for Oligonucleotide Functionalization.

The synthesis and incorporation of a tosylated phosphoramidite linker containing a disulfide bond is described. Incorporation of the linker into short DNA and RNA oligomers proceeded efficiently using automated solid phase synthesis. Treatment of the support bound oligonucleotide followed by cleavage from the solid support provided a variety of common functional handles, expanding the strategies of bifunctional modification of synthetic oligonucleotides for conjugation applications.

Arabinonucleic acids containing C5-propynyl modifications form stable hybrid duplexes with RNA that are efficiently degraded by E. coli RNase H.

The promise of the antisense approach to treat a variety of diseases with oligonucleotides and solutions to challenges that have been encountered in their development is attributable to chemical modification of the nucleic acid scaffold. Herein, we describe preliminary data regarding the synthesis of a novel C5-propynyl-ß-d-arabinouridine (araUP) phosphoramidite and its incorporation into oligonucleotides. Substitution of araUP in dT18 results in minor stabilization of duplexes formed with RNA when modifications are placed consecutively and a uniformly modified araUP 18-mer increases stability by 34 °C relative to DNA. The modified oligomer exhibits improved nuclease and serum stability when compared to DNA and duplexes formed between RNA and araUP oligonucleotides are substrates for E. coli RNase H. These preliminary results merit further investigation into C5-propynyl modified arabino nucleic acids for potential therapeutic gene silencing applications.

Recent Advances of DNA Tetrahedra for Therapeutic Delivery and Biosensing.

The chemical and self-assembly properties of nucleic acids make them ideal for the construction of discrete structures and stimuli-responsive devices for a diverse array of applications. Amongst the various three-dimensional assemblies, DNA tetrahedra are of particular interest, as these structures have been shown to be readily taken up by the cell, by the process of caveolin-mediated endocytosis, without the need for transfection agents. Moreover, these structures can be readily modified with a diverse range of pendant groups to confer greater functionality. This minireview highlights recent advances related to applications of this interesting DNA structure including the delivery of therapeutic agents ranging from small molecules to oligonucleotides in addition to its use for sensing and imaging various species within the cell.

Dual-Location Dual-Acid/Glutathione-Degradable Cationic Micelleplexes through Hydrophobic Modification for Enhanced Gene Silencing.

Gene therapy holds great promise for the treatment of acquired genetic disorders such as cancer with reduced side effects compared to chemotherapy. For gene therapy to be successful, it is crucial to develop efficient and nontoxic gene carriers to overcome the poor in vivo stability and low cellular uptake of nucleic acid-based therapeutic agents. Here, we report a new and versatile approach exploring a combination of hydrophobic modifications and dual-stimuli-responsive degradation (SRD) for controlled gene delivery with amphiphilic block copolymer-based nanocarriers. The block copolymer, synthesized by atom transfer radical polymerization, is designed with an acid-labile acetal linkage at the block junction and a pendant disulfide group in the hydrophobic block. The incorporation of labile linkages enables both disulfide-core-cross-linking and dual-location dual-acid/reduction-responsive degradation (DL-DSRD). Furthermore, the disulfide linkages integrated as hydrophobic moieties facilitate the nucleic acids to condense into nanometer-sized micelleplexes through electrostatic interactions of pendant dimethylamino groups with the anionic phosphate groups of the nucleic acids. Our preliminary results demonstrate that the DL-DSRD approach through hydrophobic modification is a robust platform in the development of gene delivery systems with enhanced colloidal stability, reduced cytotoxicity, and improved gene transfection efficiency.