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The phosphates in the backbones of DNA and RNA are often drawn like crosses but are in fact tetrahedral. Sulfur is sometimes substituted for one of the phosphate oxygens during development of nucleotide-based drugs. Because of the geometry, this swap can lead to two different isomers. Knouse et al. report a pair of phosphorus reagents that conveniently produce either isomer selectively. This ability depended on the configuration of appended limonene substituents that are subsequently jettisoned. In addition to simplifying the route to sulfur-substituted oligonucleotides, these reagents will enable more precise studies of each isomer's distinct bioactivity. Science , this issue p. 1234 Limonene-substituted phosphorus reagents offer a simplified and stereoselective route to nucleotide-based drug candidates. Phosphorothioate nucleotides have emerged as powerful pharmacological substitutes of their native phosphodiester analogs with important translational applications in antisense oligonucleotide (ASO) therapeutics and cyclic dinucleotide (CDN) synthesis. Stereocontrolled installation of this chiral motif has long been hampered by the systemic use of phosphorus(III) [P(III)]–based reagent systems as the sole practical means of oligonucleotide assembly. A fundamentally different approach is described herein: the invention of a P(V)-based reagent platform for programmable, traceless, diastereoselective phosphorus-sulfur incorporation. The power of this reagent system is demonstrated through the robust and stereocontrolled synthesis of various nucleotidic architectures, including ASOs and CDNs, via an efficient, inexpensive, and operationally simple protocol.

Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the antisense methodology because of the nuclease resistance of the phosphorothioate linkage in comparison with that of the phosphate linkage. However, as experimental data accumulated, it was detected that this chemical modification also facilitates cellular uptake and bioavailibity in vivo. Thus, today the majority of therapeutic oligonucleotides contain this modification. This review will discuss the historical development of this modification and present some of its chemical properties where they differ from those of the phosphate group. The antisense application will be discussed in the original context with cleavage of the target mRNA, but other target RNAs such as microRNAs and long noncoding RNAs will also be covered. It continues with applications where the target RNA should not be cleaved. A brief presentation of decoy oligonucleotides will be included, as well as some miscellaneous applications. Cellular uptake is a crucial step for oligonucleotides to reach their target and will be briefly reviewed. Lastly, a most surprising recent observation is the presence of phosphorothioate groups in bacterial DNA where functions still remain to be fully determined.

The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&PS DNA and restored through light-triggered chemical bond breakage of PC&PS DNA. The universality of DNACas is demonstrated by adopting the PC&PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances. CRISPR systems are powerful tools for gene editing and diagnostics, but their regulation is challenging. Here, the authors present DNACas, a light-controlled method using photocleavable phosphorothioate DNA to modulate CRISPR activity, enabling precise gene editing and one-pot diagnostic detection.

The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2′- O -methoxyethyl (2′-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1–dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2′- O -methyl (2′-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles. Chemical modification of PS-ASO therapeutics reduces binding to cellular proteins and decreases toxic side-effects.

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of R p and S p diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many R p and S p diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in R p configuration, we developed a universal s eparation system for p hosphorothioate o ligonucleotide i somers using immobilized S BD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, R p isomers of the mixture can be completely bound by SBD and separated from S p 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 R p 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 R p and S p stereoisomers, respectively. Furthermore, as each R p phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform S p configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBD Ped , 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. Graphical Abstract

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.

The development of recombinant antibody binders against phosphorothioate-modified antisense oligonucleotides (PS-ASOs) remains challenging due to the highly polyanionic character and structural flexibility of the phosphorothioate backbone. Here, we applied a next-generation sequencing (NGS)-guided phage display strategy to determine whether selection against PS-modified ASOs induces reproducible repertoire remodeling and the emergence of shared CDR3 physicochemical signatures associated with PS-ASO recognition. Two independent two-round phage display biopanning strategies against biotinylated PS-ASO targets were coupled to targeted amplicon sequencing of VH and VL FR3-CDR3-FR4 regions on the Illumina MiSeq platform. CDR3 clonotypes were reconstructed using a dedicated bioinformatic pipeline including quality filtering, read merging, in-frame translation, clonotype counting, CPM normalization, enrichment analysis, and physicochemical descriptor profiling. Representative enriched scFv clones were further evaluated by ELISA-based binding and competition assays and by fluorescence microscopy in ASO-treated cells. Deep sequencing revealed a marked reduction in repertoire diversity from Round 1 to Round 2, associated with reproducible clonal dominance across independent selection strategies. These changes were already evident at early stages of selection. A shared enriched set of 113 CDR3-VH clonotypes was identified and displayed a defined physicochemical profile, including increased positive charge, recurrent aromatic residue patterns, constrained CDR3-VH length distribution, higher theoretical pI, and reduced hydrophobicity. Representative functional assays further supported the relevance of this signature: among the selected recombinant scFv clones, 12F2 showed preferential binding to both PS1-ASO and PS2-ASO, with reduced reactivity toward the phosphodiester-backbone oligonucleotide used as control. In ASO-treated cells, 12F2 produced a detectable intracellular signal after PS-ASO transfection, whereas PO-ASO-treated cells showed absent or nearly absent signal. These results define an NGS-guided framework for identifying early-stage repertoire focusing and physicochemical signatures associated with recognition of modified nucleic acid backbones. The common property-level features suggest convergent binding solutions compatible with recognition of phosphorothioate-associated molecular features, supporting the rational prioritization of candidate binders against challenging polyanionic targets.

Phosphorothioate (PT) DNA modifications in E. coli and Salmonella are turned over to maintain bacterial fitness through decreased susceptibility to genomic instability caused by hypochlorous-acid-mediated PT oxidation. Genomic modification by sulfur in the form of phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, PT sulfur has redox and nucleophilic properties that suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. In experiments involving isotopic labeling coupled with mass spectrometry, we observed sulfur replacement in PTs at a rate of ∼2% h −1 in unstressed Escherichia coli and Salmonella enterica . Whereas PT levels were unaffected by exposure to hydrogen peroxide (H 2 O 2 ) or hypochlorous acid (HOCl), PT turnover increased to 3.8–10% h −1 after HOCl treatment and was unchanged by H 2 O 2 , consistent with the repair of HOCl-induced sulfur damage. PT-dependent sensitivity to HOCl extended to cytotoxicity and DNA strand breaks, which occurred at HOCl doses that were orders of magnitude lower than the corresponding doses of H 2 O 2 . The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during infections in humans.

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

A scalable chemical method to control phosphorothioate chirality demonstrates its impact on the efficacy of antisense oligonucleotides. Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that S p-configured PS linkages are stabilized relative to R p, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3′- S p S p R p, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.