Refining LNA safety profile by controlling phosphorothioate stereochemistry

Funder, Erik Daa; Albæk, Nanna; Moisan, Annie; Sewing, Sabine; Koch, Troels

doi:10.1371/journal.pone.0232603

Cited by 13 publications

(14 citation statements)

References 32 publications

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“…Phosphorothioate (PS) modification of (oligo)nucleotides is one of the most frequently used synthetic approaches for investigating nucleic acid metabolism or for improving the pharmacokinetics of therapeutic or diagnostic molecules. − Many applications of thio-modified oligonucleotides, such as the elucidation of ion-binding sites on RNA and identification of phosphate sites important for ligand or protein binding or for catalysis, − assume that this modification has little or no effect on the secondary structure of nucleic acids. However, it has been shown that incorporation of the PS moiety alters the negative charge distribution and can significantly alter the conformation of nucleic acids, thus changing their biological activity. , Furthermore, substitution of a nonbridging oxygen in the phosphate residue with sulfur usually generates an additional stereogenic center at the phosphorus atom yielding a pair of diastereomers, , which are often differentially recognized by specific proteins or enzymes. − Interestingly, a natural PS backbone modification was shown to be an epigenetic mark in bacterial DNA, − and recently it was also found in both prokaryotic and eukaryotic rRNAs …”

Section: Introductionmentioning

confidence: 99%

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect

et al. 2021

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mRNA-based therapies and vaccines constitute a disruptive technology with the potential to revolutionize modern medicine. Chemically modified 5′ cap structures have provided access to mRNAs with superior translational properties that could benefit the currently flourishing mRNA field. Prime examples of compounds that enhance mRNA properties are antireverse cap analog diastereomers that contain an O-to-S substitution within the β-phosphate (β-S-ARCA D1 and D2), where D1 is used in clinically investigated mRNA vaccines. The compounds were previously found to have high affinity for eukaryotic translation initiation factor 4E (eIF4E) and augment translation in vitro and in vivo . However, the molecular basis for the beneficial “thio-effect” remains unclear. Here, we employed multiple biophysical techniques and captured 11 cap analog-eIF4E crystallographic structures to investigate the consequences of the β-O-to-S or -Se substitution on the interaction with eIF4E. We determined the S P / R P configurations of β-S-ARCA and related compounds and obtained structural insights into the binding. Unexpectedly, in both stereoisomers, the β-S/Se atom occupies the same binding cavity between Lys162 and Arg157, indicating that the key driving force for complex stabilization is the interaction of negatively charged S/Se with positively charged amino acids. This was observed for all structural variants of the cap and required significantly different conformations of the triphosphate for each diastereomer. This finding explains why both β-S-ARCA diastereomers have higher affinity for eIF4E than unmodified caps. Binding affinities determined for di-, tri-, and oligonucleotide cap analogs suggested that the “thio-effect” was preserved in longer RNAs. Our observations broaden the understanding of thiophosphate biochemistry and enable the rational design of translationally active mRNAs and eIF4E-targeting drugs.

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Section: Introductionmentioning

confidence: 99%

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect

et al. 2021

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“…Hence, strategies using oligonucleotides with a limited number of PS linkages have gained traction, presumably as such oligonucleotides have fewer/weaker protein interactions [104]. One additional concern with PS modifications is the introduction of stereoisomerism to the structure, which may also be a complicating factor in toxicity [105]. The ZEN end-modified anti-miRs with 2 0 -OME modifications avoid such stereoisomeric complexity, confer nuclease resistance and appear relatively safe [72], [71], and perhaps offer a more straightforward route to robust stereodefined anti-miRs over PS alternatives.…”

Section: Discussionmentioning

confidence: 99%

Capturing the RNA castle: Exploiting MicroRNA inhibition for wound healing

et al. 2021

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The growing pipelines of RNA-based therapies herald new opportunities to deliver better patient outcomes for complex disorders such as chronic nonhealing wounds associated with diabetes. Members of the microRNA (miRNA) family of small noncoding RNAs have emerged as targets for diverse elements of cutaneous wound repair, and both miRNA enhancement with mimics or inhibition with antisense oligonucleotides represent tractable approaches for miRNA-directed wound healing. In this review, we focus on miRNA inhibition strategies to stimulate skin repair given advances in chemical modifications to enhance the performance of antisense miRNA (anti-miRs). We first explore miRNAs whose inhibition in keratinocytes promotes keratinocyte migration, an essential part of reepithelialisation during wound repair. We then focus on miRNAs that can be targeted for inhibition in endothelial cells to promote neovascularisation for wound healing in the context of diabetic mouse models. The picture that emerges is that direct comparisons of different anti-miRNAs modifications are required to establish the most translationally viable options in the chronic wound environment, that direct comparisons of the impact of inhibition of different miRNAs are needed to quantify and rank their relative efficacies in promoting wound repair, and that a standardised human ex vivo model of the diabetic wound is needed to reduce reliance on mouse models that do not necessarily enhance mechanistic understanding of miRNA-targeted wound healing.

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“…LNA amidites were dissolved at 0.1 M in MeCN. Synthesis was performed as described previously [ 20 ].…”

Section: Methodsmentioning

confidence: 99%

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers

Kielpinski

Funder

Schmidt

et al. 2021

Nucleic Acid Therapeutics

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Phosphorothioate (PS) modification of antisense oligonucleotides (ASOs) is a critical factor enabling their therapeutic use. Standard chemical synthesis incorporates this group in a stereorandom manner; however, significant effort was made over the years to establish and characterize the impact of chiral control. In this work, we present our in-depth characterization of interactions between Escherichia coli RNase H and RNA-DNA heteroduplexes carrying chirally defined PS groups. First, using a massive parallel assay, we showed that at least a single R p-PS group is necessary for efficient RNase H-mediated cleavage. We followed by demonstrating that this group needs to be aligned to the phosphate-binding pocket of RNase H, and that chiral status of other PS groups in close proximity to RNase H does not affect cleavage efficiency. We have shown that RNase H's PS chiral preference can be utilized to guide cleavage to a specific chemical bond. Finally, we present a strategy for ASO optimization by mapping preferred RNase H cleavage sites of a non-thioated compound, followed by introduction of R p-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an S p-PS at the same location.

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Refining LNA safety profile by controlling phosphorothioate stereochemistry

Cited by 13 publications

References 32 publications

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect

Structural Insights into the Interaction of Clinically Relevant Phosphorothioate mRNA Cap Analogs with Translation Initiation Factor 4E Reveal Stabilization via Electrostatic Thio-Effect

Capturing the RNA castle: Exploiting MicroRNA inhibition for wound healing

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers

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