Phosphorothioate Modification Research Articles

Nanopore Sequencing for Detection and Characterization of Phosphorothioate Modifications in Native DNA Sequences.

Bacterial DNA is subject to various modifications involved in gene regulation and defense against bacteriophage attacks. Phosphorothioate (PT) modifications are protective modifications in which the non-bridging oxygen in the DNA phosphate backbone is replaced with a sulfur atom. Here, we expand third-generation sequencing techniques to allow for the sequence-specific mapping of DNA modifications by demonstrating the application of Oxford Nanopore Technologies (ONT) and the ELIGOS software package for site-specific detection and characterization of PT modifications. The ONT/ELIGOS platform accurately detected PT modifications in a plasmid carrying synthetic PT modifications. Subsequently, studies were extended to the genome-wide mapping of PT modifications in the Salmonella enterica genomes within the wild-type strain and strains lacking the PT regulatory gene dndB (ΔdndB) or the PT synthetic gene dndC (ΔdndC). PT site-specific signatures were observed in the established motifs of GAAC/GTTC. The PT site locations were in close agreement with PT sites previously identified using the Nick-seq technique. Compared to the wild-type strain, the number of PT modifications are 1.8-fold higher in ΔdndB and 25-fold lower in ΔdndC, again consistent with known regulation of the dnd operon. These results demonstrate the suitability of the ONT platform for accurate detection and identification of the unusual PT backbone modifications in native genome sequences.

Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.

Involvement of the DNA Phosphorothioation System in TorR Binding and Anaerobic TMAO Respiration in Salmonella enterica.

ABSTRACTAlthough the phosphorothioate (PT) modification, in which the nonbridging oxygen in the DNA sugar-phosphate backbone is replaced by sulfur, has been reported to play versatile roles in multiple cellular processes, very little data have been obtained to define the role of PT in epigenetic regulation. In this study, we report that the PT system in Salmonella enterica serovar Cerro 87 is involved in the transcriptional regulation of the torCAD operon encoding the trimethylamine N-oxide (TMAO) respiration machinery that enables the use of TMAO as a terminal electron acceptor for respiration when oxygen is not available. In vitro, PT enhanced the binding of the transcriptional activator of the torCAD operon, namely, TorR, to its DNA substrate (tor boxes). However, in vivo, the PT modification protein complex DndCDE downregulated torCAD transcription through competing with the binding of TorR to the tor boxes. The altered expression of torCAD caused by PT modification proteins affected cell growth that relied on TMAO respiration. To our knowledge, this is the first report supporting that PT proteins participate in transcriptional regulation, showing a new function of PT systems.

Molecular Structure of Single-Stranded DNA on the ZnS Surface of Quantum Dots.

DNA-based nanoparticle assemblies have emerged as leading candidates in the development of bioimaging materials, photonic devices, and computing materials. Here, we combine atomistic simulations and experiments to characterize the wrapping mechanism of chimeric single-stranded DNA (ssDNA) on CdSe-ZnS (core-shell) quantum dots (QDs) at different ratios of the phosphorothioate (PS) modification of the bases. We use an implicit solvent, all-atom ssDNA model to match the experimentally calculated ssDNA conformation at low salt concentrations. Through simulation, we find that 3-mercaptopropionic acid (MPA) induces electrostatic repulsion and O-(2-mercaptoethyl)-Ó-methyl-hexa (ethylene glycol) (mPEG) induces steric exclusion, and both reduce the binding affinity of ssDNA. In both simulation and experiment, we find that ssDNA is closer to the QD surface when the QD size is larger. The effect of the PS-base ratio on the conformation of ssDNA is also elaborated in this work. We found through MD simulations, and confirmed by transmission electron microscopy, that the maximum valence numbers are 1, 2, and 3 on QDs of 6, 9, and 14 nm in diameter, respectively. We conclude that the maximum ssDNA valence number is linearly related to the QD size, n ∝ R, and justify this finding through an electrostatic repulsion mechanism.

Novel Naturally Occurring Dipeptides and Single-Stranded Oligonucleotide Act as Entry Inhibitors and Exhibit a Strong Synergistic Anti-HIV-1 Profile.

IntroductionThe availability of new classes of antiretroviral drugs is critical for treatment-experienced patients due to drug resistance to and unwanted side effects from current drugs. Our aim was therefore to evaluate the anti-HIV-1 activity of a new set of antivirals, dipeptides (WG-am or VQ-am) combined with a single-stranded oligonucleotide (ssON). The dipeptides were identified as naturally occurring and enriched in feces and systemic circulation in HIV-1-infected elite controllers and were proposed to act as entry inhibitors by binding to HIV-1 gp120. The ssON is DNA 35-mer, stabilized by phosphorothioate modifications, which acts on the endocytic step by binding to cell host receptors and inhibiting viruses through interference with binding to nucleolin.MethodsChou–Talalay’s Combination Index method for quantifying synergism was used to evaluate the drug combinations. Patient-derived chimeric viruses encoding the gp120 (env region) were produced by transient transfection and used to evaluate the antiviral profile of the combinations by drug susceptibility assays.ResultsWe found that the combination WG-am:ssON or VQ-am:ssON had low combination index values, suggesting strong antiviral synergism. Of the two combinations, WG-am:ssON (1 mM:1 μM) had high efficacy against all prototype or patient-derived HIV-1 isolates tested, independent of subtype including the HIV-1-A6 sub-subtype. In addition, the antiviral effect was independent of co-receptor usage in patient-derived strains.ConclusionWG-am and ssON alone significantly inhibited HIV-1 replication regardless of viral subtype and co-receptor usage, and the combination WG-am:ssON (1 mM:1 μM) was even more effective due to synergism.

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides.

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)‐rich and phosphorothioate (PS)‐modified DNA oligonucleotide was investigated using two fragmentation techniques: trap‐type collision‐induced dissociation (“CID”) and beam‐type CID also termed as higher‐energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC‐MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T‐rich DNA oligonucleotides. Specifically, HCD provided more sequence‐defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS‐modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T‐rich PS‐modified DNA oligonucleotides.

Physicochemical property evaluation of modified oligonucleotides by traveling-wave ion mobility mass spectrometry.

Therapeutic oligonucleotides have molecular weights of more than 6000 Da. They typically contain chemically modified structures such as phosphorothioate (PS) and a locked nucleic acid (LNA). To determine the effect of the length and chemical modification on the physicochemical properties, various nucleic acids with different lengths and modified structures were analyzed using traveling-wave ion mobility mass spectrometry (TWIMS). The physicochemical characteristics of the modified oligonucleotides were determined using IM-MS. Each oligonucleotide was evaluated by confirming the multivalent charge state drift times, collision cross-section (CCS) values, and CCS widths. By plotting the m/z for oligonucleotides of different lengths and the CCS values at each charge state, a bottoming-out shape plot at one charge per 4.0-3.5 bases was confirmed. Moreover, significant differences were observed in the CCS values between the PS-modified and unmodified oligonucleotides. The PS-modified oligonucleotide showed a wider CCS range that was proportional to the PS modification ratio of the oligonucleotide sequence. The TWIMS results showed a correlation between the length and modification of oligonucleotides and the CCS values. In addition, it suggested that each charge state of the oligonucleotide ion has different physicochemical properties.

Structural Fingerprinting of siRNA Therapeutics by Solution NMR Spectroscopy.

Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat a wide variety of illnesses, including diseases where traditional drug development efforts have failed. To date, there are 14 short oligonucleotide therapeutics and 2 messenger RNA (mRNA) vaccines approved by the U.S. Food and Drug Administration (FDA), which demonstrates the potential of nucleic acids as a platform for the development of safe and effective medicines and vaccines. Despite the increasing popularity of nucleic acid-based drugs, there has been a paucity of high-resolution structural techniques applied to rigorously characterize these molecules during drug development. Here, we present application of nuclear magnetic resonance (NMR) methods to structurally "fingerprint" short oligonucleotide therapeutics at natural isotope abundance under full formulation conditions. The NMR methods described herein leverage signals arising from the native structural features of nucleic acids, including imino, aromatic, and ribose resonances, in addition to non-native chemistries, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and phosphorothioate (PS) modifications, introduced during drug development. We demonstrate the utility of the NMR methods to structurally "fingerprint" a model short interfering RNA (siRNA) and a sample that simulated the drug product Givosiran. We anticipate broad applicability of the NMR methods to other nucleic acid-based therapeutics due to the generalized nature of the approach and ability to monitor many quality attributes simultaneously.

Ultraviolet Photodissociation and Activated Electron Photodetachment Mass Spectrometry for Top-Down Sequencing of Modified Oligoribonucleotides.

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.

Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-SRp substitution is inhibitory in Mg2+, thiophilic Cd2+ rescues HHRz activity. Mn2+ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-SRp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn2+ and Cd2+ coordination to Rp and Sp phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd2+ does indeed bind to S in the thio-substituted ligand, Mn2+ coordinates to the non‑sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of MnO over MnS coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn2+ coordination preference of oxygen in either Rp or Sp A9 phosphorothioates differentially tunes catalytic activity, with MnO coordination in the A9-SRp phosphorothioate enzyme being inhibitory.

Sensing Metal Ions with Phosphorothioate-Modified DNAzymes.

Phosphorothioate (PS) modification refers to replacing one of the nonbridging oxygen atoms in nucleic acids with sulfur. PS modifications can be easily introduced during solid-phase DNA synthesis. It has been extensively used in ribozyme and DNAzyme research to achieve a bioinorganic understanding of metal binding, bioanalytical applications of metal detection, and chemical biology of DNA modification. It allows for the access of new chemistry, not available to natural DNA. Since each PS modification is accompanied by the production of a chiral phosphorus center, a key technical challenge is to separate the two diastereomers called Rp and Sp. In this chapter, we describe our methods of HPLC-based separation followed by ligation to generate a long and fluorescently modified DNAzyme substrate. Subsequently, the use of the modified substrate for activity assay to understand metal binding and for metal ion detection is also described.

Phosphorothioate modification improves exon-skipping of antisense oligonucleotides based on sulfonyl phosphoramidates in mdx mouse myotubes.

2'-O-Methyl (2'-OMe) antisense oligonucleotides (AOs) possessing a various number of 4-(trimethylammonio)butylsulfonyl or tosyl phosphoramidates (N+ and Ts-modifications, respectively) instead of a native phosphodiester linkage were designed to skip exon-23 in dystrophin pre-mRNA transcript in mdx mice myotubes. AOs bearing several zwitterionic N+ modifications in the sequence had remarkably increased thermal stability towards complementary mRNA in comparison with 2'-OMe-RNAs having negatively charged Ts and phosphorothioate (PS) linkages. However, only Ts-modified AOs exhibited a similar level of exon skipping in comparison with fully modified PS-containing 2'-OMe-RNA, whereas the exon skipping induced by N+ modified AOs was much lower with no exon-skipping detected for AOs having seven N+ modifications. The level of exon-skipping was improved once Ts and especially N+ moieties were used in combination with PS-modification, most likely through improved cellular and nuclear uptake of AOs. These results provide new insights on expanding the design of novel chemically modified AOs based on phosphate modifications.

Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides.

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers.

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 Rp-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 Rp-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an Sp-PS at the same location.

Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses.

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT‐containing diribonucleotides with native species in RNA hydrolysates by high‐resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT‐specific iodine‐desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′‐O‐methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.

Site-Specific Scissors Based on Myeloperoxidase for Phosphorothioate DNA.

The tool box of site-specific cleavage for nucleic acid has been an increasingly attractive subject. Especially, the recent emergence of the orthogonally activatable DNA device is closely related to the site-specific scission. However, most of these cleavage strategies are based on exogenous assistance, such as laser irradiation. Endogenous strategies are highly desirable for the orthogonally regulatable DNA machine to explore the crucial intracellular biological process and cell signal network. Here, we found that the accurate site-specific cleavage reaction of phosphorothioate (PT) modified DNA by using myeloperoxidase (MPO). A scissors-like mechanism by which MPO breaks PT modification through chloride oxidation has been revealed. Furthermore, we have successfully applied the scissors to activate PT-modified hairpin-DNA machines to produce horseradish peroxidase (HRP)-mimicking DNAzyme or initiate hybridization chain reaction (HCR) amplification. Since MPO plays an important role in the pathway related to oxidative stress in cells, through the HCR amplification activated by this tool box, the oxidative stress in living cells has been robustly imaged. This work proposes an accurate and endogenous site-specific cleavage tool for the research of biostimuli and the construction of DNA molecular devices.

Pharmacodynamic and Pharmacokinetic Properties of Full Phosphorothioate Small Interfering RNAs for Gene Silencing In Vivo

State-of-the-art small interfering RNA (siRNA) therapeutics such as givosiran and fitusiran are constructed from three variable components: a fully-modified RNA core that conveys metabolic stability, a targeting moiety that mediates target-cell uptake, and a linker. This structural complexity poses challenges for metabolite characterization and risk assessment after long-term patient exposure. In this study, we show that basic phosphorothioate modification of a siRNA targeting the oncoprotein Lin28B provides a useful increase in metabolic stability, without greatly compromising potency. We found that its stability in vitro matched that of nanoparticle-free patisiran in serum and surpassed it in liver tritosome extracts, although it did not reach the stability of the fitusiran siRNA core structure. Liver and kidney were the main sites of accumulation after its subcutaneous administration in mice. Despite the lack of a delivery agent-free antitumor effect, we anticipate our study to be a starting point to develop alternative siRNA scaffolds that can be degraded into naturally-occurring metabolites and help alleviate the aforementioned challenges. Furthermore, Lin28B is a promising target for cancers, and the development of such simplified siRNA analogs, possibly together with novel targeting units, holds potential.

An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction.Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection.Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

SspABCD-SspFGH Constitutes a New Type of DNA Phosphorothioate-Based Bacterial Defense System.

Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.IMPORTANCE We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system. To our surprise, ssDNA PT modification can also pair with a newly identified 3-gene sspFGH cassette to fend off phage infection with a different mode of action from that of SspE. Interestingly, both SspFGH and SspE can pair with the same SspABCD module for antiphage defense, and their combination provides Escherichia coli JM109 with additive phage resistance up to 105-fold compared to that for either barrier alone. This agrees with our observation that SspFGH and SspE coexist in 36 bacterial genomes, highlighting the diversity of the gene contents and molecular mechanisms of PT-based defense systems.