Phosphorothioate Oligonucleotides Research Articles

Selectivity limits of and opportunities for ion pair chromatographic separation of oligonucleotides

Here it was investigated how oligonucleotide retention and selectivity factors are affected by electrostatic and non-electrostatic interactions in ion pair chromatography. A framework was derived describing how selectivity depends on the electrostatic potential generated by the ion-pair reagent concentration, co-solvent volume fraction, charge difference between the analytes, and temperature. Isocratic experiments verified that, in separation problems concerning oligonucleotides of different charges, selectivity increases with increasing surface potential and analyte charge difference and with decreasing co-solvent volume fraction and temperature. For analytes of the same charge, for example, diastereomers of phosphorothioated oligonucleotides, selectivity can be increased by decreasing the co-solvent volume fraction or the temperature and has only a minor dependency on the ion-pairing reagent concentration. An important observation is that oligonucleotide retention is driven predominantly by electrostatic interaction generated by the adsorption of the ion-pairing reagent. We therefore compared classical gradient elution in which the co-solvent volume fraction increases over time versus gradient elution with a constant co-solvent volume fraction but with decreasing ion-pair reagent concentration over time. Both modes decrease the electrostatic potential. Oligonucleotide selectivity was found to increase with decreasing ion-pairing reagent concentration. The two elution modes were finally applied to two different model antisense oligonucleotide separation problems, and it was shown that the ion-pair reagent gradient increases the selectivity of non-charge–based separation problems while maintaining charge-difference–based selectivity.

Mitigation of analyte loss on metal surfaces in liquid chromatography

The adsorptive loss of acidic analytes in liquid chromatography was investigated using metal frits. Repetitive injections of acidic small molecules or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were observed for adenosine 5′-(α,β-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the positive charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were saturated with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solutions. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid organic/inorganic surface technology were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quantitative recovery of a 15–35 mer oligodeoxythymidine mixture was achieved using column hardware modified with hybrid surface technology, without a need for column conditioning prior to analysis.

Evaluation of micropillar array columns for chromatographic separation of phosphorothioated oligonucleotides and their diastereomers.

Chromatographic analysis of therapeutic oligonucleotides is challenging due to the presence of closely related impurities, degradants or metabolites and due to the presence of phosphorothioate bonds, which introduce chiral centers. In the present study, ion pair reversed phase chromatography of oligonucleotides on micropillar array columns was investigated. Two commonly used mobile phase conditions were included. With 16.3mM triethylamine and 400mM hexafluoroisopropanol, the separation of 16-mer oligonucleotides differing in the number and positions of phosphorothioate linkages as well as some n-1 and n-2 truncations demonstrated complete suppression of diastereoselectivity. Although the positional phosphorothioate isomers evaluated could not be resolved, an increase in phosphorothioate bonds resulted in more retention. A therapeutic 19-mer RNA sequence with 2'-fluor and 2'-O-methyl modifications showed partial separation of some very close impurities. When using 15mM triethyl ammonium acetate in the mobile phase, diastereomer selectivity was clearly observed for all analytes. The best result was obtained for the 19-mer RNA therapeutic mimic with four phosphorothioate bonds, since all 16 theoretical diastereomers were clearly observed under the conditions tested. A limited benchmark exercise demonstrated the improved capability of the new micropillar array columns. Therefore, these columns can be positioned as a valuable alternative when challenging oligonucleotide separations are expected.

Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Liquid chromatography-mass spectrometry has been widely implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs). In this study, we developed a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ion-pairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis. HILIC separation of mixtures of unmodified and fully phosphorothioate-modified DNA oligonucleotides and their synthetic 3’ exonuclease-digested metabolites were also assessed. High-resolution mass spectrometric (HRMS) analysis was used to determine the deconvoluted masses of oligonucleotide and siRNA standards and their impurities. To enable unbiased sequence characterization with tandem mass spectrometry (MS/MS), we also optimized higher-energy C-trap dissociation (HCD) on improving the sequence coverage of DNA and RNA oligonucleotides. Lastly, we evaluated on-column sensitivity for a phosphorothioate oligonucleotide by performing targeted analysis with either targeted selected ion monitoring (tSIM) or parallel reaction monitoring (PRM). Higher on-column sensitivity of 13 ng, equivalent to 2.0 pmol, of a phosphorothioate oligonucleotide was achieved by tSIM analysis as compared to PRM analysis.

History of Research on Antisense Oligonucleotide Analogs

In the search for novel therapeutics, antisense oligonucleotide (ASO) analogs have been a major focus of research for over 40 years. They use the antisense strategy, namely they have a nucleic acid base sequence that is complementary to a portion of a specific mRNA that is produced in the cell, or to a viral RNA, in order to selectively inhibit gene expression. Oligonucleotides need to be chemically modified to stabilize them against hydrolysis by endogenous nucleases. Until now several phosphorothioate (PS) oligonucleotide analogs have been approved by the FDA for human use. This article seeks to provide a history of this subject to date.

Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins.

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2′-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein–protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein—dephosphorylation of deoxynucleotide triphosphates (dNTPs)—implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3’ to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

Stereochemistry of internucleoside phosphorus atom affects sugar pucker and acid hydrolysis of N3′-P5′ thio-phosphoramidates

Investigation of the sugar pucker of the two diastereomers of dinucleotide N3′-P5′-thio-phosphoramidates TNPSTNH2 and TNPSTOH shows that the Sp isomer adopts a more C3′-endo (North) sugar ring configuration than the Rp counterpart. In contrast, P-stereochemistry of oligonucleotide phosphorothioate (O3′-P5′) compounds has no effect on the nucleoside sugar puckering. This difference is also reflected in the different rate of acid hydrolysis for the two isomers. Thus, the Rp stereoisomer with less prevalent C3′-endo configuration has an acid hydrolysis rate constant ~50% higher than that of the Sp molecule. The TNPOTNH2 and TNPOTOH dinucleotides are hydrolyzed an order of magnitude faster than TNPSTNH2 and TNPSTOH, respectively. In addition, dinucleotides with the terminal 3′-OH group are hydrolyzed two times faster than their 3′-NH2 counterparts.

Expanding the Scope of the Cleavable N-(methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates.

Oligonucleotides modified by a 2′-deoxy-2′-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ‘‘switched off-state’’. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-β-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a β-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19–47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to β-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T0.5 ca 7 h) to hydrazone-based structures.

Antisense oligonucleotide p45Skp-2 suppresses migratory chemotactic and metastasis of oral malignant Burkitt’s lymphoma cell through down-regulation of MTA-1 and induction of E-cadherin mechanism

Introduction: Burkitt’s lymphoma is a high-grade B-cell neoplasm and one of the most aggressive malignancies of lymphoid origins which found mainly in the paediatric population. The treatment options of this tumour are still limited. However, a new strategy for refractory tumour, phosphorothioate oligonucleotide antisense technique has watched with keen interest. This study was aimed to examine the effect of antisense p45Skp-2 (Skp-2 AS) suppressed migratory chemotactic and metastasis of oral malignant Burkitt’s lymphoma (Raji) cell through down-regulation of MTA-1 and E-cadherin. Methods: True experiment laboratory with post-test control group design was confirmed in this study. The efficiency of Skp-2 AS in the suppression of cell chemotactic migration was examined by Boyden chamber assay. To evaluate the inhibition of cell metastasis was conducted by decreasing MTA-1 expression protein. The expressions of MTA-1, E-cadherin and α-tubulin protein were investigated by Western blot analysis. Results: The results revealed that the number of chemotactic migration of Skp-2 AS treated Raji cell was significantly decreased when compared with that of sense p45Skp-2 (Skp-2 S) and scrambled control (SC) cells (P<0.05) followed by decreased expressions of MTA-1 protein and overexpression of E-cadherin. Interestingly, the expression of α-tubulin protein as an internal control was approximately similar in each transfectant cells. Conclusion: p45Skp-2 have an antitumor activity via suppression of migratory chemotactic activity and metastasis on oral Burkitt’s lymphoma cells through down-regulation of MTA-1 and induction of E-cadherin proteins targeting this molecule could represent a promising new therapeutic approach for this type of cancer.

Approaches for Systemic Delivery of Dystrophin Antisense Peptide Nucleic Acid in the mdx Mouse Model.

Antisense-mediated exon skipping constitutes a promising new modality for treatment of Duchenne Muscular Dystrophy (DMD), which is caused by gene mutations that typically introduce a translation stop codon in the dystrophin gene, thereby abolishing production of functional dystrophin protein. The exon removal can restore translation to produce a shortened, but still partially functional dystrophin protein. Peptide nucleic acid (PNA) as a potential antisense drug has previously been shown to restore the expression of functional dystrophin by splice modulation in the mdx mouse model of DMD. In this study, we compare systemic administration of a 20-mer splice switching antisense PNA oligomer through intravenous (i.v.) and subcutaneous (s.c.) routes in the mdx mice. Furthermore, the effect of in situ forming depot technology (BEPO®) and PNA-oligonucleotide formulation was studied. In vivo fluorescence imaging analysis showed fast renal/bladder excretion of the PNA (t½ ∼ 20 min) for i.v. administration, while s.c. administration showed a two to three times slower excretion. The release from the BEPO depot exhibited biphasic kinetics with a slow release (t½ ∼ 10 days) of 50% of the dose. In all cases, some accumulation in kidneys and liver could be detected. Formulation of PNA as a duplex hybridization complex with a complementary phosphorothioate oligonucleotide increased the solubility of the PNA. However, none of these alternative administration methods resulted in significantly improved antisense activity. Therefore, either more sophisticated formulations such as designed nanoparticles or conjugation to delivery ligands must be utilized to improve both pharmacokinetics as well as tissue targeting and availability. On the other hand, the results show that s.c. and BEPO depot administration of PNA are feasible and allow easier, higher, and less frequent dosing, as well as more controlled release, which can be exploited both for animal model studies as well as eventually in the clinic in terms of dosing optimization.

SARS-CoV-2 will constantly sweep its tracks: a vaccine containing CpG motifs in 'lasso' for the multi-faced virus.

During the current COVID-19 pandemic, the global ratio between the dead and the survivors is approximately 1 to 10, which has put humanity on high alert and provided strong motivation for the intensive search for vaccines and drugs. It is already clear that if we follow the most likely scenario, which is similar to that used to create seasonal influenza vaccines, then we will need to develop improved vaccine formulas every year to control the spread of the new, highly mutable coronavirus SARS-CoV-2. In this article, using well-known RNA viruses (HIV, influenza viruses, HCV) as examples, we consider the main successes and failures in creating primarily highly effective vaccines. The experience accumulated dealing with the biology of zoonotic RNA viruses suggests that the fight against COVID-19 will be difficult and lengthy. The most effective vaccines against SARS-CoV-2 will be those able to form highly effective memory cells for both humoral (memory B cells) and cellular (cross-reactive antiviral memory T cells) immunity. Unfortunately, RNA viruses constantly sweep their tracks and perhaps one of the most promising solutions in the fight against the COVID-19 pandemic is the creation of 'universal' vaccines based on conservative SARS-CoV-2 genome sequences (antigen-presenting) and unmethylated CpG dinucleotides (adjuvant) in the composition of the phosphorothioate backbone of single-stranded DNA oligonucleotides (ODN), which can be effective for long periods of use. Here, we propose a SARS-CoV-2 vaccine based on a lasso-like phosphorothioate oligonucleotide construction containing CpG motifs and the antigen-presenting unique ACG-containing genome sequence of SARS-CoV-2. We found that CpG dinucleotides are the most rare dinucleotides in the genomes of SARS-CoV-2 and other known human coronaviruses, and hypothesized that their higher frequency could be responsible for the unwanted increased lethality to the host, causing a ‘cytokine storm’ in people who overexpress cytokines through the activation of specific Toll-like receptors in a manner similar to TLR9-CpG ODN interactions. Interestingly, the virus strains sequenced in China (Wuhan) in February 2020 contained on average one CpG dinucleotide more in their genome than the later strains from the USA (New York) sequenced in May 2020. Obviously, during the first steps of the microevolution of SARS-CoV-2 in the human population, natural selection tends to select viral genomes containing fewer CpG motifs that do not trigger a strong innate immune response, so the infected person has moderate symptoms and spreads SARS-CoV-2 more readily. However, in our opinion, unmethylated CpG dinucleotides are also capable of preparing the host immune system for the coronavirus infection and should be present in SARS-CoV-2 vaccines as strong adjuvants.

Refining LNA safety profile by controlling phosphorothioate stereochemistry.

Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.

Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase

Oligonucleotide therapeutics have contributed remarkably to healthcare, being well suited for the treatment of intractable diseases that are difficult to approach using conventional drug modalities. However, as common techniques of oligonucleotide analysis rely on reversed-phase or ion-exchange liquid chromatography and thus employ toxic organic solvents and/or ion-pairing reagents, better alternatives are highly sought after. Poly(N-isopropylacrylamide) (PNIPAAm) is widely used in temperature-responsive chromatography (TRC), which relies on column temperature variation to control the physical properties of the stationary phase and, unlike conventional reversed-phase liquid chromatography, avoids the use of toxic organic solvents and complicated gradient methods. Herein, PNIPAAm copolymer hydrogel-modified silica beads were used for the simultaneous analysis of multiple synthetic oligonucleotides by TRC to recognize differences in the length of single nucleotides, single bases, and the number of phosphorothioated sites. Temperature-responsive elution was observed in all cases. Each separation of all combinations of multiple oligonucleotides was better at higher temperatures above the lower critical solution temperature and was performed without the use of organic solvents and gradient methods. In the case of multiply phosphorothioated oligonucleotides, good separation was achieved using an aqueous solvent and isocratic elution in the absence of ion-pairing reagents. Thus, the developed procedure was concluded to be well suited for oligonucleotide analysis.Graphical abstract

Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup.

Determination of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liquid chromatography (IPRP) with a mobile phase containing triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatography (AEX) is an orthogonal separation mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liquid chromatography(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liquid chromatography (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop volume and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further separation of additional impurities.

Development of an ICP-MS/MS approach for absolute quantification and determination of phosphodiester to phosphorothioate ratio in therapeutic oligonucleotides

A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phosphorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both are considered as critical quality attributes and should be determined using robust validated methods. The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur) and optionally determination of sodium (or any other metal) as a counter ion. The performance of the method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model substance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision.

Hydrophilic interaction in solid-phase extraction of antisense oligonucleotides.

The presented studies aimed to develop a new and simple extraction method based on hydrophilic interaction for antisense oligonucleotides with different modifications. For this purpose, solid-phase extraction cartridges with unmodified silica were used. All extraction steps were performed by utilizing water, acetonitrile, acetone or their mixtures. The results obtained show that a high content (95%) of organic solvent, used during sample loading, is critical to achieve a successful extraction, while elution with pure water allows effective oligonucleotides desorption. The recovery values were greater than 90% in the case of unmodified DNA, phosphorothioate, 2'-O-(2-methoxyethyl) and 2'-O-methyl oligonucleotides. For the mixture of phosphorothioate oligonucleotide and its two synthetic metabolites, the recovery values for the standard solutions were in the range of 70-75%, while for spiked human plasma, 45-50%. The developed method is simple, may be performed in a short time and requires simple solvents like water or acetonitrile/acetone, thus showing promise as an alternative to chaotropic salt-based or ion pair-based SPE methods.

Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents

Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 5–20mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.

In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3′ thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3′ and 5′ thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks.In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency.In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides.