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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.

R-loops are DNA–RNA hybrids enriched at CpG islands (CGIs) that can regulate chromatin states 1 – 8 . How R-loops are recognized and interpreted by specific epigenetic readers is unknown. Here we show that GADD45A (growth arrest and DNA damage protein 45A) binds directly to R-loops and mediates local DNA demethylation by recruiting TET1 (ten-eleven translocation 1). Studying the tumor suppressor TCF21 (ref. 9 ), we find that antisense long noncoding (lncRNA) TARID (TCF21 antisense RNA inducing promoter demethylation) forms an R-loop at the TCF21 promoter. Binding of GADD45A to the R-loop triggers local DNA demethylation and TCF21 expression. TARID transcription, R-loop formation, DNA demethylation, and TCF21 expression proceed sequentially during the cell cycle. Oxidized DNA demethylation intermediates are enriched at genomic R-loops and their levels increase upon RNase H1 depletion. Genomic profiling in embryonic stem cells identifies thousands of R-loop-dependent TET1 binding sites at CGIs. We propose that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs. GADD45A directly binds to R-loops and mediates local DNA demethylation by recruiting TET1. lncRNA-mediated formation of R-loops at the promoter of the tumor suppressor TCF21 triggers GADD45A binding, demethylation and expression.

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.

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery. The exosome is a ribonucleolytic complex that plays part in RNA processing and degradation. Here, the authors reveal an RNA clearance event in homologous recombination and show the function of EXOSC10, a component of the exosome, in the homeostasis of RNA molecules synthesized at DSB sites.

Circular intronic RNAs (ciRNAs) escaping from DBR1 debranching of intron lariats are co-transcriptionally produced from pre-mRNA splicing, but their turnover and mechanism of action have remained elusive. We report that RNase H1 degrades a subgroup of ciRNAs in human cells. Many ciRNAs contain high GC% and tend to form DNA:RNA hybrids (R-loops) for RNase H1 cleavage, a process that appears to promote Pol II transcriptional elongation at ciRNA-producing loci. One ciRNA, ciankrd52 , shows a stronger ability of R-loop formation than that of its cognate pre-mRNA by maintaining a locally open RNA structure in vitro . This allows the release of pre-mRNA from R-loops by ci-ankrd52 replacement and subsequent ciRNA removal via RNase H1 for efficient transcriptional elongation. We propose that such an R-loop dependent ciRNA degradation likely represents a mechanism that on one hand limits ciRNA accumulation by recruiting RNase H1 and on the other hand resolves R-loops for transcriptional elongation at some GC-rich ciRNA-producing loci.

Low activity has been the primary obstacle impeding the use of DNA enzymes (DNAzymes) as gene silencing agents in clinical applications. Here we describe the chemical evolution of a DNAzyme with strong catalytic activity under near physiological conditions. The enzyme achieves ~65 turnovers in 30 minutes, a feat only previously witnessed by the unmodified parent sequence under forcing conditions of elevated Mg 2+ and pH. Structural constraints imposed by the chemical modifications drive catalysis toward a highly preferred U GU D motif (cut site underlined) that was validated by positive and negative predictions. Biochemical assays support an autonomous RNA cleavage mechanism independent of RNase H1 engagement. Consistent with its strong catalytic activity, the enzyme exhibits persistent allele-specific knock-down of an endogenous mRNA encoding an undruggable oncogenic KRAS target. Together, these results demonstrate that chemical evolution offers a powerful approach for discovering new chemotype combinations that can imbue DNAzymes with the physicochemical properties necessary to support therapeutic applications. Low activity currently prevents the wider use of DNA enzymes (DNAzymes). Here the authors report the chemical evolution of a DNAzyme with high catalytic activity under near physiological conditions: the enzyme achieves ~65 turnovers in 30 minutes.

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations. Long non coding RNA TERRA transcripts can form R-loops at chromosome ends. Here, the authors reveal a role for the helicase RTEL in affecting TERRA levels and localization.

Catalysis by members of the RNase H superfamily of enzymes is generally believed to require only two Mg2+ ions that are coordinated by active-site carboxylates. By examining the catalytic process of Bacillus halodurans RNase H1 in crystallo, however, we found that the two canonical Mg2+ ions and an additional K+ failed to align the nucleophilic water for RNA cleavage. Substrate alignment and product formation required a second K+ and a third Mg2+, which replaced the first K+ and departed immediately after cleavage. A third transient Mg2+ has also been observed for DNA synthesis, but in that case it coordinates the leaving group instead of the nucleophile as in the case of the RNase H1 hydrolysis reaction. These transient cations have no contact with the enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and strand-transfer reactions in a single active site may similarly require cation trafficking coordinated by the substrate.

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3'-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.