Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Development, validation and application of an ion-pair reversed-phase liquid chromatography-tandem mass spectrometry method for the quantification of nusinersen.

Background: The fully phosphorothioate-modified oligonucleotide (OGN) nusinersen has low ionization efficiency in the negative ion mode, resulting in a low mass spectrometry response. There have been no relevant reports on developing a LC-MS method for the determination of nusinersen by optimizing mobile phase composition. Materials & methods: Mobile phase additives comprised of 15 mM triethylamine/25 mM 1,1,1,3,3,3-hexafluoro-2-propanol with a pH of 9.6. Nusinersen was extracted from plasma using Oasis® HLB solid-phase extraction (Waters, MA, USA). Results & conclusion: By adjusting the pH of the mobile phase to 9.6 by optimizing the type and concentration of ion-pair reagents, a high mass spectrometry response was obtained. The developed method was applied to nusinersen and met the requirements for the pharmacokinetic study of nusinersen in rabbits.

Hydrophilic interaction liquid chromatography with mass spectrometry for the separation and identification of antisense oligonucleotides impurities and nusinersen metabolites.

With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza.

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry.

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication.

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA-DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.

Methods to functionalize gold nanoparticles with tandem-phosphorothioate DNA: role of physicochemical properties of the phosphorothioate backbone in DNA adsorption to gold nanoparticles.

Perception of the differences in the physicochemical properties of phosphorothioate DNA (PS-DNA) and phosphodiester DNA (PO-DNA) greatly aids in understanding the AuNP-DNA binding process. Replacing one non-bridging oxygen atom of the anionic phosphodiester backbone with a sulfur atom leads to a major change in the DNA adsorption mechanism of AuNPs. In this work, we investigated and compared salt-aging, low pH-assisted, and freeze-thaw methods for conjugating phosphorothioate-modified oligonucleotides to AuNPs. The results obtained clearly demonstrate that only the pH-assisted method can successfully bind tandem phosphorothioate DNA to gold nanoparticles and sufficiently maintain the colloidal stability of AuNPs. When a phosphate group is converted to a phosphorothioate group, the negative charge of the phosphate group is located on the sulfur atom. Due to the soft nature of sulfur (a very weak H-bond acceptor), the negative charge on the sulfur atom cannot be shielded even with the gradual addition of salt to increase the ionic strength, so, the pH-assisted based method is the best for the functionalization of AuNPs with tandem-PS DNA.

Stereo-Controlled Liquid Phase Synthesis of Phosphorothioate Oligonucleotides on a Soluble Support.

5'-O-(2-Methoxyisopropyl) (MIP)-protected 2'-deoxynucleosides as chiral P(V)-building blocks, based on the limonene-derived oxathiaphospholane sulfide, were synthesized and used for the assembly of di-, tri-, and tetranucleotide phosphorothioates on a tetrapodal pentaerythritol-derived soluble support. The synthesis cycle consisted of two reactions and two precipitations: (1) the coupling under basic conditions, followed by neutralization and precipitation and (2) an acid catalyzed 5'-O-deacetalization, followed by neutralization and precipitation. The simple P(V) chemistry together with the facile 5'-O-MIP deprotection proved efficient in the liquid phase oligonucleotide synthesis (LPOS). Ammonolysis released nearly homogeneous Rp or Sp phosphorothioate diastereomers in ca. 80% yield/synthesis cycle.

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

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.

Systematic Analysis of Chemical Modifications of Phosphorothioate Antisense Oligonucleotides that Modulate Their Innate Immune Response.

While rare, some gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) can induce a noncanonical TLR9-dependent innate immune response. In this study, we performed systematic analyses of the roles of PS ASO backbone chemistry, 2' modifications, and sequence in PS ASO induced TLR9 signaling. We found that each of these factors can contribute to altering PS ASO induced TLR9 signaling, and in some cases the effects are quite dramatic. We also found that the positioning (5' vs. 3') of a particular backbone or 2' modification within a PS ASO can affect its TLR9 signaling. Interestingly, medicinal chemical strategies that decrease TLR9 signaling for one sequence can have opposing effects on another sequence. Our results demonstrate that TLR9 signaling is highly PS ASO sequence dependent, the mechanism of which remains unknown. Despite this, we determined that placement of two mesyl phosphoramidate linkages within the PS ASO gap is the most promising strategy to mitigate PS ASO dependent TLR9 activation to enhance the therapeutic index and, therefore, further streamline PS ASO drug development.

Assessment of the Effect of Organ Impairment on the Pharmacokinetics of 2'-MOE and Phosphorothioate Modified Antisense Oligonucleotides.

The pharmacokinetics (PK) of 2'-O-methoxyethyl and phosphorothioate antisense oligonucleotides (ASOs), with or without N-acetyl galactosamine conjugation, have been well characterized following subcutaneous or intravenous drug administration. However, the effect of organ impairment on ASO PK, primarily hepatic or renal impairment, has not yet been reported. ASOs distribute extensively to the liver and kidneys, where they are metabolized slowly by endo- and exonucleases, with minimal renal excretion as parent drug (<1%-3%). This short review evaluated the effect of organ impairment on ASO PK using 3 case studies: (1) a phase 1 renal impairment study evaluating a N-acetyl galactosamine-conjugated ASO in healthy study participants and study participants with moderate renal impairment, (2) a phase 2 study evaluating an unconjugated ASO in patients with end-stage renal disease; and (3) a phase 3 study evaluating an unconjugated ASO, which included patients with mild hepatic or renal impairment. Results showed that patients with end-stage renal disease had a mild increase (≈34%) in total plasma exposure, whereas mild or moderate renal impairment showed no effect on plasma PK. The effect of hepatic impairment on ASO PK could not be fully evaluated due to lack of data in moderate and severe hepatic impairment study participants. Nonetheless, available data suggest that mild hepatic impairment had no effect on ASO exposure.

SIDT2 Inhibits Phosphorothioate Antisense Oligonucleotide Activity by Regulating Cellular Localization of Lysosomes.

Phosphorothioate (PS)-modified antisense oligonucleotide (ASO) drugs enter cells through endocytic pathways where a majority are entrapped within membrane-bound endosomes and lysosomes, representing a limiting step for antisense activity. While late endosomes have been identified as a major site for productive PS-ASO release, how lysosomes regulate PS-ASO activity beyond macromolecule degradation remains not fully understood. In this study, we reported that SID1 transmembrane family, member 2 (SIDT2), a lysosome transmembrane protein, can robustly regulate PS-ASO activity. We showed that SIDT2 is required for the proper colocalization between PS-ASO and lysosomes, suggesting an important role of SIDT2 in the entrapment of PS-ASOs in lysosomes. Mechanistically, we revealed that SIDT2 regulates lysosome cellular location. Lysosome location is largely determined by its movement along microtubules. Interestingly, we also observed an enrichment of proteins involved in microtubule function among SIDT2-binding proteins, suggesting that SIDT2 regulates lysosome location via its interaction with microtubule-related proteins. Overall, our data suggest that lysosome protein SIDT2 inhibits PS-ASO activity potentially through its interaction with microtubule-related proteins to place lysosomes at perinuclear regions, thus, facilitating PS-ASO's localization to lysosomes for degradation.

A simple and green solid phase extraction method for oligonucleotides using adsorbent with amino and carboxylic moieties.

The study aimed to use a material with amino and carboxylic moieties to extract unmodified and phosphorothioate oligonucleotides. The charge of amine and carboxyl groups at the surface has changed with the change in pH of the adsorption (pH 4.5) and desorption solution (pH 9.5). Thus, both the binding and elution of the oligonucleotides were based on electrostatic interactions, and the procedure required only 10 mM ammonium acetate, with the change of pH depending on the extraction step. The developed procedure was successfully applied to extract oligonucleotides from aqueous solutions and serum samples. The method is simple and fast, providing good reproducibility (SD between 1 and 4%) and relatively high oligonucleotide recovery (81-98% for standards, 60-71% for diluted serum samples, and 80-92 for LLE serum extracts). Moreover, only environmentally friendly solvents were used.

Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

Phosphorothioate-linked guanine/cytosine-based stem-loop oligonucleotides induce the extracellular release of mitochondrial DNA from peritoneal B1a cells.

It has been previously demonstrated that phosphorothioate-linked GpC-based stem-loop oligonucleotides (GC-SL ODN) induce the release of mitochondrial DNA (mtDNA) from chronic lymphocytic leukemia (CLL) B cells. Although CLL B cells are believed to originate from CD5+ B cells because of their phenotypic similarities, it remains unclear whether GC-SL ODN can stimulate CD5+ B1 cells to secrete mtDNA. To explore this possibility, we compared the frequency of the mtDNA-producing population among peritoneal cells after GC-SL ODN treatment. We found that mtDNA-releasing cells are enriched for peritoneal CD19+ B cells upon GC-SL ODN challenge. Among peritoneal CD19+ B cells, the CD5+ B1a subpopulation was a primary cellular source of mtDNA secretion in GC-SL ODN-elicited immune responses. GC-SL ODN-stimulated mtDNA release by B1a cells was positively regulated by MyD88 and TRIF signaling pathways. In vivo GC-SL ODN treatment increased lipopolysaccharide-induced activation of innate immune cells such as NK cells, suggesting the immune-enhancing effects of mtDNA secretion. Furthermore, the loop size formed by GC-SL ODNs was a critical factor in inducing mtDNA release by B1a cells. Taken together, our results identified GC-SL ODN as promising biomaterials for enhancing immune responses.

Separation of phosphorothioate oligonucleotide impurities by WAX HPLC under high organic content elution conditions.

The separation of impurities in phosphorothioate diester (PS) oligonucleotides is complicated by (1) the presence of a very large number of diastereoisomers, e.g., 219 for a 20-mer oligonucleotide, (2) peak broadening due to the hydrophobic character of the sulfur atom, and (3) the chemical similarity of the impurities to the parent oligonucleotide and each other. Further difficulties arise due to the chemical nature of oligonucleotides, which display a complex mixture of ionic, hydrophobic, H-bonding, and other functionalities. To minimize hydrophobic interactions and peak broadening due to the PS modification, we have developed a novel method that combines a weak anion exchange (WAX) column with a mobile phase elution system designed to maximize separation by a single ionic/electrostatic interaction. We found that although chaotropes are helpful, the most significant beneficial effect of the hydrophilic WAX column is that high-organic, low-salt mobile phase is required for product elution. Separations are also benefitted by pH gradient effects on stationary phase electrostatic potential and analyte ionization. An extraordinary degree of separation is achieved by the new WAX method in comparison to SAX (strong anion exchange) chromatography. For the first time, the extent of deamination of PS oligonucleotides is directly determined by a chromatography-only method. The approach, representative results, and the mechanisms of separation are discussed.

Phosphorothioate oligonucleotides separation in ion-pairing reversed-phase liquid chromatography: effect of temperature.

Analysis of diastereomers of phosphorothioate oligonucleotides in ion-pairing reversed-phase liquid chromatography is affected not only by the character and concentration of ion-pairing system, but also by the separation temperature. In this work, eight ion-pairing systems at two concentrations buffered with acetic acid were used with octadecyl column to investigate the effects of temperature (in the range from 20 °C to 90 °C) on retention, diastereomeric separation, resolution of mers of different length and resolution of oligonucleotides with different number of phosphorothioate linkages. It was observed that elevated temperature suppresses the diastereomeric separation and oligonucleotide peaks become narrower. This improves the resolution of n and n-1 mers at elevated temperature. Plots of ln k (k = retention factor) versus reciprocal absolute temperature show that for 100 mM ion-pairing systems the increase in temperature does not lead to simple decrease in oligonucleotides retention as generally observed in reversed-phase liquid chromatography. The aim of this work is to improve chromatographic method for analysis of phosphorothioate oligonucleotides.

Insights into innate immune activation via PS-ASO-protein-TLR9 interactions.

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary-in part-as a function of 2'modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO-protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

NAT10 and DDX21 Proteins Interact with RNase H1 and Affect the Performance of Phosphorothioate Oligonucleotides.

RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.

An LNA-amide modification that enhances the cell uptake and activity of phosphorothioate exon-skipping oligonucleotides.

Oligonucleotides that target mRNA have great promise as therapeutic agents for life-threatening conditions but suffer from poor bioavailability, hence high cost. As currently untreatable diseases come within the reach of oligonucleotide therapies, new analogues are urgently needed to address this. With this in mind we describe reduced-charge oligonucleotides containing artificial LNA-amide linkages with improved gymnotic cell uptake, RNA affinity, stability and potency. To construct such oligonucleotides, five LNA-amide monomers (A, T, C, 5mC and G), where the 3'-OH is replaced by an ethanoic acid group, are synthesised in good yield and used in solid-phase oligonucleotide synthesis to form amide linkages with high efficiency. The artificial backbone causes minimal structural deviation to the DNA:RNA duplex. These studies indicate that splice-switching oligonucleotides containing LNA-amide linkages and phosphorothioates display improved activity relative to oligonucleotides lacking amides, highlighting the therapeutic potential of this technology.