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Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

ABSTRACT: Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.

SUBMITTER: Genna V

PROVIDER: S-EPMC10250214 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

Genna Vito V Iglesias-Fernández Javier J Reyes-Fraile Laura L Villegas Nuria N Guckian Kevin K Seth Punit P Wan Brad B Cabrero Cristina C Terrazas Montserrat M Brun-Heath Isabelle I González Carlos C Sciabola Simone S Villalobos Anabella A Orozco Modesto M

Nucleic acids research 20230601 10

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic met ...[more]

PMID: 37099382

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Project description:Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.

Project description:Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60-90% of the starting Rp isomers, with PS linkage not locating at 5' or 3' end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method. KEY POINTS: • SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC. • SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers. • SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme.

Dataset Information

Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

Publications

Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

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