Host RNA N6-methyladenosine and incoming DNA N6-methyldeoxyadenosine modifications cooperatively elevate the condensation potential of DNA to activate immune surveillance

Na Wang; Qiaoling Liu; Bo Wang; Zhuo Yang; Siru Li; Ran Li; Xinyuan Liang; Jiayu Fan; Hui Wang; Zhen Sun; Ling Dong; Yueru Hou; Shengnan Wang; Chengli Song; Yang Wang; Chunshan Quan; Qingkai Yang; Lina Wang

doi:10.1016/j.ymthe.2024.10.027

. 2024 Oct 28;32(12):4418–4434. doi: 10.1016/j.ymthe.2024.10.027

Host RNA N⁶-methyladenosine and incoming DNA N⁶-methyldeoxyadenosine modifications cooperatively elevate the condensation potential of DNA to activate immune surveillance

Na Wang ^1,⁵, Qiaoling Liu ^1,⁵, Bo Wang ^2,⁵, Zhuo Yang ^3,⁵, Siru Li ¹, Ran Li ¹, Xinyuan Liang ¹, Jiayu Fan ¹, Hui Wang ¹, Zhen Sun ¹, Ling Dong ¹, Yueru Hou ¹, Shengnan Wang ¹, Chengli Song ¹, Yang Wang ¹, Chunshan Quan ⁴, Qingkai Yang ^1,^∗, Lina Wang ^1,^∗∗

PMCID: PMC11638879 PMID: 39473181

Abstract

Self-non-self discrimination is fundamental to life, thereby even microbes can apply DNA modifications to recognize non-self DNA. However, mammalian cytosolic DNA sensors indiscriminately bind DNA, necessitating specific mechanism(s) for self-non-self discrimination. Here, we show that mammalian RNA N⁶-methyladenosine (m6A) and incoming DNA N⁶-methyldeoxyadenosine (6mdA) cooperatively elevate the condensation potential of DNA to activate immunosurveillance. RNA m6A modification was found to enhance the activation of cyclic guanosine monophosphate-AMP synthase (cGAS) via increasing DNA phase separation. And 6mdA further increased the phase separation potential of DNA. Consistently, host RNA m6A and incoming DNA 6mdA modifications cooperatively elevated the incoming DNA condensation and cGAS activation. Moreover, we developed a prodrug, QKY-613. QKY-613 promoted a discriminative incorporation of 6mdA into viral DNAs to elevate host immune surveillance, and decreased mortality in virus-infected aged mice. Our results link nucleic acid modification diversity with immune surveillance via phase separation, which might be targeted for therapeutic intervention.

Keywords: N⁶-methyladenosine, N⁶-methyldeoxyadenosine, cyclic GMP-AMP synthase, phase separation, DNA sensor, immune surveillance

Graphical abstract

Wang and colleagues show that host RNA m6A and incoming DNA 6mdA modifications cooperatively elevate the incoming DNA condensation and cGAS activation, providing a mechanism for self-non-self discrimination.

Introduction

Self-non-self discrimination is the fundament of life.¹ Mammals are evolved to acquire cytosolic DNA sensors including cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) to detect the non-self DNA (incoming DNA).² As a central DNA sensor, cGAS binds DNA and synthesizes cyclic GMP-AMP (cGAMP) to mount immune responses including the production of interferon (IFN).³^,⁴ However, cGAS indiscriminately bind the phosphate backbones, but not the bases of DNA,⁵^,⁶ necessitating specific mechanism(s) to facilitate self-non-self recognition. Because an inevitable challenge for virus is to package its DNA into tiny virion, condensation is a universal feature of viral DNA (a classical non-self DNA). And the structure of cGAS-DNA complex raises a role for DNA condensation in self-non-self discrimination. To activate cGAS, every single cGAS molecule needs to bind two condensed dsDNA strands to form the cGAS2n-DNA2 (n ≥ 1) complex (Figure 1A).⁵^,⁶^,⁷ DNA molecules are negatively charged, and it is energetically unfavorable to condense DNA particularly at low DNA concentrations. Consistently, DNA condensation (phase separation) is pivotal to activate cGAS.⁸

RNA m6A elevates intracellular condensation of incoming DNA and cGAS activation

(A) Schematic showing that, in the cGAS-DNA complex (PDB: 5N6I), one cGAS molecule (blue) binds two condensed dsDNA strands (red). cGAS-DNA complex model was visualized by PyMOL using surface representations. (B) Schematic showing that host RNA m6A and incoming DNA 6mdA modifications cooperatively elevate the cytosolic condensation potential of incoming DNA to activate cGAS-mediated immune responses including the IFN production. (C) IFNb mRNA levels in the wildtype (WT) and FTO KO cells infected with Adv at a multiplicity of infection (MOI) of 1 for 6 h using vehicle as control (Ctrl). IFNb mRNA levels were assessed using real-time qRT-PCR. In this study, all data are shown as mean ± standard deviation, and error bars indicate standard deviations. Unless specifically noted, p values were from unpaired two-tailed Student’s t-test (∗∗p < 0.01, ∗∗∗p < 0.001). n = 6. (D) ELISA analyses of the IFNb protein levels from the WT and FTO KO MEF or MLF cells infected with 1 MOI Adv for 12 h using vehicle as Ctrl. n = 3. (E) MS analyses of mRNA m6A levels in the MEF cells treated with vehicle (mock), 5 μM UZH1a (UZH), or 50 μM m6A nucleoside (m6A) for 12 h. n = 6. (F and G) IFNb mRNA (F) and protein levels (G) in the m6A-pretreated MEF cells. Cells were treated with vehicle, 5 μM UZH1a or 50 μM m6A nucleoside for 12 h, then infected with 1 MOI Adv for 6 h (F) (n = 6) and 12 h (G) (n = 3). (H and I) IFNb mRNA (H) and protein levels (I) in the m6A-pretreated WT and cGAS KO MEF cells treated with 10 μM RU.521 (RU) and Adv. Cells were treated with vehicle, 5 μM UZH1a, or 50 μM m6A nucleoside for 12 h. After 10 μM RU.521 treatment for 2 h, the resultant cells were infected with 1 MOI Adv for 6 h (H) (n = 6) and 12 h (I) (n = 3). (J and K) IFNb mRNA (J) and protein levels (K) in the m6A-pretreated MEF cells. Cells were treated with vehicle, 5 μM UZH1a or 50 μM m6A nucleoside for 12 h, then transfected with 200 ng/mL DNA for 2 h (J) (n = 6) and 12 h (K) (n = 3). (L and M) IFNb mRNA (L) and protein levels (M) in the m6A-pretreated WT and cGAS KO MEF cells treated with 10 μM RU.521 and DNA transfection. Cells were treated with vehicle, 5 μM UZH1a, or 50 μM m6A nucleoside for 12 h. After 10 μM RU.521 treatment for 2 h, the resultant cells were transfected with 200 ng/mL DNA for 2 h (L) (n = 6) and 12 h (M) (n = 3). (N) Representative fluorescent images of MEF cells transfected with 200 ng/mL DNA for 30 min. Green: fluorescein 5-isothiocyanate (FITC)-labeled DNA; blue: 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. (O) DNA condensate folds in (N). The number in Mock was taken as 1. n = 6.

Base modifications of nucleic acids play critical roles in both self-non-self discrimination and condensation of nucleic acids. Mammalian DNAs have only one abundant modification, 5-methyldeoxycytidine (5mdC).⁹ Conversely, microbial DNAs have diverse modifications, prevalently DNA N⁶-methyldeoxyadenosine (6mdA).⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴ In microbes, DNA 6mdA can serve as a marker for self-non-self discrimination systems such as the well-known restriction-modification system.¹⁵ 6mdA levels in microbes and lower eukaryotes are more than 10,000-fold higher than theses in mammals.⁹^,¹⁰^,¹¹ Interestingly, mammals show low DNA but high RNA modification diversity, and mammalian RNA N⁶-methyladenosine (m6A) is the most abundant mammalian RNA modification.¹⁶ However, little is known about the significance of these diversity imbalances. Notably, m6A RNA and m6A-binding proteins are involved in the condensation (phase separation) of nucleic acids,¹⁷^,¹⁸^,¹⁹^,²⁰ which is critical to the non-self nucleic acid sensing.¹ Moreover, both m6A-binding proteins and incoming DNA-cGAS condensates are frequently localized in stress granules (SGs).¹⁷^,¹⁸^,²¹^,²²^,²³ These phenomena are further emphasized by the studies showing that m6A-binding proteins can bind DNA-RNA hybrid.²⁴^,²⁵ These observations suggest a role for RNA m6A and DNA 6mdA modifications in the detection of incoming DNA.

Moreover, a substantial challenge to global health is that the high mutation rates of viruses almost inevitably lead to impairing/evading the host immune surveillance, particularly in older adults. But, how to discriminatively elevate the immune surveillance against non-self nucleic acids is less understood. A central mechanism for the high mutation of viruses is the low fidelity of their nucleotide polymerases,²⁶ which frequently leads to the incorporation of incorrect nucleotides into their genomes. Based on the low fidelity of viral nucleotide polymerase, nucleotide analogues (such as anti-Covid-19 drug, Remdesivir) can be incorporated into viral genome to inhibit its elongation.²⁷ Unfortunately, due to the high mutation rate of virus, the resistance to these virus-targeting strategies is almost inevitable,²⁶ necessitating the development of new strategies.

In this study, we show that host RNA m6A and incoming DNA 6mdA modifications cooperatively elevate the DNA condensation potential to activate immune surveillance (Figure 1B). And we demonstrate that a designed compound, QKY-613, can promote a discriminative incorporation of 6mdA into viral DNAs to elevate host immune surveillance, and decreased mortality in virus-infected aged mice. These results link the nucleic acid modification diversity and immune surveillance via phase separation (condensation), which might be targeted for intervention. Our study also suggests that mammals evolved DNA and RNA modification diversities to facilitate self-non-self discrimination, providing a potential explanation for the diversity imbalances of nucleic acid modifications.

Results

RNA m6A elevates intracellular condensation of incoming DNA and cGAS activation

During our previous study about RNA m6A,²⁸ adenovirus (Adv) was used to restore the gene expression in mice. Interestingly, ELISAs indicated that knockout (KO) of fat mass and obesity-associated protein (FTO)²⁹ resulted in more IFNb production in response to acute viral infection (Figure S1A). Consistent with the in vivo observations, Adv infection also led to more IFNb production in FTO KO mouse embryonic fibroblast (MEF) and mouse lung fibroblast (MLF) cells (Figures 1C, 1D, and S1B). To further evaluate the role of m6A, m6A nucleoside and an inhibitor of methyltransferase 3, UZH1a,³⁰ were applied to increase and decrease intracellular RNA m6A levels, respectively (Figures 1E, S1C, and S1D). And m6A nucleoside was also used to rescued the RNA m6A levels in the cells treated with UZH1a (Figure 1E). As shown in Figures 1F and 1G, m6A inhibition (UZH1a) decreased the IFNb production, while m6A supplementation (m6A nucleoside) increased IFNb production in response to Adv infection. It suggested that the overall RNA m6A levels increased the immune response. Because Adv is a DNA virus and cGAS is central to the DNA-sensing,³^,³¹ cGAS inhibitor (RU.521) and cGAS KO MEF cells were used to evaluate the role of cGAS in the m6A-increased IFNb production. RU.521 effectively inhibited cGAS activity, but showed little impact on the cGAS protein levels (Figure S1E).³² As shown in Figures 1H and 1I, cGAS inhibition or KO abrogated the m6A-increased IFNb production, indicating that cGAS mediated the m6A-increased IFNb production.

To exclude the potential influence of viral components other than DNA, 45 base pair (bp) DNA was transfected into the MEF cells. To assess the transfection efficiency, the DNA was labeled with fluorescein 5-isothiocyanate-fluorophore. Flow cytometry analyses indicated that m6A inhibition and supplementation showed no significant effect on the transfection efficiency (Figure S1F). Consistent with the results of DNA viral infection, m6A inhibition reduced the potential of incoming DNA (transfected DNA) to stimulate the IFNb production, while m6A supplementation increased the IFNb production (Figures 1J and 1K). The cGAS inhibition or KO abrogated the m6A-increased productions of cGAMP (Figures S1G and S1H) and IFNb (Figures 1L and 1M). In line with the results in MEF cells, similar results were also observed in the MLF cells (Figures S1I–S1N).

Interestingly, we noticed that m6A inhibition decreased the condensation (phase separation) potential of incoming DNA, while m6A supplementation enhanced the condensation potential of DNA (Figures 1N and 1O). Due to the central role of DNA condensation in cGAS activation, these results suggested that m6A-RNA might increase the cGAS activation via enhancing the incoming DNA condensation.

Colocalization and condensation analyses suggested the candidate genes for m6A RNA-increased condensation of incoming DNA

We then evaluated the impact of m6A-RNA on intracellular cGAS-DNA condensation. Consistent with a previous study,⁸ immunofluorescence (IF) analyses showed that cGAS and incoming DNA were co-localized, and they formed the cGAS-DNA condensates in the MEF cells transfected with DNA (Figure 2A). M6A inhibition decreased the cGAS-DNA condensation, while m6A supplementation elevated the cGAS-DNA condensation (Figures 2A and S2A), supporting that m6A enhances cGAS-DNA condensation to increase the cGAS activation. Notably, as previously described,²¹ DNA condensates were co-localized with SGs marked by G3BP SG assembly factor 1 (G3BP1) (Figures 2B and S2B), leading to the remarkable SG-cGAS-DNA colocalization and condensation (Figure 2C). Consistently, further analyses indicated that m6A enhanced the SG-cGAS-DNA colocalization and condensation (Figures 2C and S2C).

Since SGs are well known to enrich m6A-RNA and m6A-binding proteins,¹⁷^,¹⁸ cGAS- and m6A-binding proteins in SGs might play a role in the m6A-increased DNA-condensation and cGAS activation (Figure 2D). Meta-analyses were then preformed to identify the candidate genes (Table S1). As shown in Figure 2E, 24 candidate proteins located in SGs displayed both m6A- and cGAS-binding potentials. Condensation prediction analyses indicated that 18 proteins showed condensation potential (Figure S2D). Among these 18 proteins, 8 proteins (IGF2BP1, YBX3, ILF3, CPSF7, CAPRIN1, YTHDF1, YTHDF2, and YTHDF3) were located in cytoplasm (Figures 2F and S2D). The notable enrichment of YTHDF family suggested a role for YTHDF1, YTHDF2, and YTHDF3 (hereafter denoted YF1, YF2, and YF3, respectively) in m6A-increased DNA condensation. This was further emphasized by the reported interaction between YF1–3 and DNA-RNA hybrid.²⁴^,²⁵ We then evaluated the colocalization between YF and cytosolic cGAS-DNA condensates. IF analyses indicated that YF1, YF2, and YF3 were co-localized with the cytosolic cGAS-DNA condensates (Figures 2G and S2E). Consistently, co-immunoprecipitation (coIP) analyses suggested that incoming DNA might form a complex with cGAS, G3BP1, YF1, YF2 and YF3. Interestingly, m6A inhibition decreased the potential of DNA to bind these proteins, while m6A supplementation enhanced the binding (Figure S2F), suggesting a role of YF1–3 in condensing DNA to activate cGAS.

M6A-RNA and YTHDFs enhance the DNA condensation and cGAS activation

To evaluate the role of YF1–3, YF1–3 were separately knocked down using small interfering RNA (siRNA) (Figure S3A). Among YF1–3, YF2 knockdown (KD) most notably decreased the m6A-increased condensation of incoming DNA (Figure S3B). Further coIP analyses indicated that YF2 bound cGAS via the C-terminal of YF2 (Figure S3C), suggesting that YFs (particularly YF2) might facilitate the cGAS-DNA condensation and cGAS activation. YF1–3 proteins were then expressed and purified in vitro (Figure S3D). To assess the impact of m6A-RNA on DNA-condensation, Alexa Fluor 568 (AF568)-labeled YF protein was incubated with the DNA and/or m6A-RNA. Turbidity (Figure 3A) and fluorescent (Figures 3B–3D, S3E, and S3F) analyses indicated that RNA increased the formation of YF-DNA condensates, while RNA m6A modification could further elevate the potential of RNA to enhance YF-DNA condensation. These observations suggested that RNA m6A modification might increase DNA-condensation at least partially via YF(s).

Next, we evaluated the impact of m6A-RNA and YFs on cGAS-DNA condensation and subsequent cGAS activation. Human cGAS protein was expressed and purified in vitro (Figure S3G). Consistent with a previous study,⁸ in the absence of RNA and YFs, the mixing of DNA and cGAS at high concentrations (≥1,000 nM) led to cGAS-DNA condensation, but cGAS at physiological concentration (≤50 nM, approximately 10 nM in HeLa cells) poorly condensed DNA (Figures 3E, 3F, and S3H). Consistently, in the absence of RNA and YFs, cGAS at physiological concentrations showed poor activity to produce cGAMP (Figure S3I), in line with a previous study.⁸ In line with the potential to increase the DNA condensation, m6A-RNA and YF2 remarkably elevated the cGAS-DNA condensation (Figure 3G) and cGAS activation (Figure 3H). To further evaluate the role of YFs, we in vitro expressed and purified the five proteins (IGF2BP1, YBX3, ILF3, CPSF7, and CAPRIN1) mentioned above (Figure S3J). As shown in Figure S3K, YF2 more notably enhanced cGAS activity than IGF2BP1, YBX3, ILF3, CPSF7, and CAPRIN1 did (Figure S3K), raising the potential of YTHDFs to increase cGAS activity.

Collectively, these results indicated that m6A-RNA and YTHDFs enhanced the cGAS-DNA condensation and cGAS activation.

RNA m6A, DNA 6mdA, and YTHDFs cooperatively elevate the in vitro DNA condensation and cGAS activation

Due to the central role of DNA-protein binding in condensation, we assessed the DNA-protein interaction using a metabolite affinity responsive target fluorescence quenching (MARTFQ) assay.²⁸ Based on the biophysical principle of metabolite (or ligand)-induced intrinsic fluorescence quenching of the target protein, MARTFQ revealed that m6A-ssRNA displayed the most potential to quench the YF2 intrinsic fluorescence, indicating that m6A-ssRNA most notably bound YF2 (Figures 4A and S4A). Interestingly, m6A-ssRNA:ssDNA hybrid also more notably bound YF2 than ssRNA:ssDNA hybrid did (Figures 4A and S4A).

Based on the similarity between m6A and 6mdA, the above observations suggested a role for DNA 6mdA modification in YF2-DNA binding. Further analyses showed that, even in the absence of RNA, 6mdA still increased the YF2-DNA binding (Figures 4B, S4B, and S4C). To exclude the potential methodological influence of the MARTFQ assay, electrophoretic mobility shift assay (EMSA) and microscale thermophoresis (MST) were applied. Based on the principle that the electrophoretic mobility of a protein-DNA complex is less than that of the free DNA, EMSA indicated that DNA 6mdA modification decreased the electrophoretic mobility, suggesting that DNA 6mdA modification elevated YF-DNA binding (Figures 4C and 4D). Among YF1–3, YF2, most notably bound 6mdA-DNA (Figure 4E). In line with the EMSA results, the 6mdA-elevated YF-DNA binding was observed in MST assays (Figures S4D and S4E), which detect changes in the hydration shell of molecules and measures biomolecule interactions under close-to-native conditions.

Next, we evaluated the impact of DNA 6mdA modification on the YF2-DNA condensation. In line with the binding results, DNA 6mdA modification increased the YF2-DNA condensation (Figure S4F). Interestingly, m6A-RNA could further enhance the potential of DNA 6mdA to increase the YF2-DNA condensation (Figures S4F and S4G). Consistently, in the presence of YF2, RNA m6A and DNA 6mdA modifications cooperatively elevated the cGAS-DNA condensation (Figure 4F) and cGAS activation (Figure 4G).

RNA 5-methylcytosine (m5C) and DNA 5mdC are abundant modifications of RNA and DNA in mammals, respectively.⁹^,³⁴ We then evaluated the impact for RNA m5C and DNA 5mdC on cGAS condensation and activity. Fluorescent analyses showed that RNA m5C and DNA 5mdC showed little impact on the cGAS-DNA condensation (Figure S4H). Consistently, RNA m5C and DNA 5mdC had poor influence on cGAS activity (Figure S4I).

Host RNA m6A and incoming DNA 6mdA modifications cooperatively elevate the intracellular incoming DNA condensation and cGAS activation

We next assessed the impact of RNA m6A and incoming DNA 6mdA modifications on the cGAS-DNA condensation and cGAS activation in the cellular context. Previous study has shown that E. coli plasmid DNA more efficiently activates cGAS than synthetic or PCR DNAs.³⁵ Considering that 6mdA is the most abundant modification in E. coli DNAs (incoming DNA),¹¹ we evaluated the impact of DNA 6mdA on the immune stimulatory potential of linear E. coli plasmid DNA (Figure 5A). To exclude the influence of DNA sequence and length, pCI-neo plasmid was used as PCR template. The plasmid and 400-bp PCR DNAs were separately digested to produce 323-bp linear DNAs (Figure 5B). Resultant digested DNAs were treated with oxidative demethylase (Alkb)³⁶ to remove 6mdA (Figures 5C, 5D, and S5A–S5C). As shown in Figure 5E, digested plasmid DNA did stimulate more IFNb production as previously described,³⁵ while deletion of 6mdA reduced the IFNb production, indicating that 6mdA elevated immune stimulatory potential of incoming DNA (E. coli DNA). Next, we investigated the role of 6mdA in the mammalian DNA via transfecting the protein of adenine methyltransferase (Dam) (Figure S5D), a DNA 6mdA-methylase in E. coli. As a DNA 6mdA-methylase, Dam modifies DNA in a sequence-dependent and species-independent manner, enabling Dam to methylate mammalian DNA.³⁷ Consistently, Dam increased both DNA 6mdA (Figures S5E and S5F) and IFNb production (Figure S5G). Given that mammals show extremely low DNA 6mdA modification, these results raise a role of 6mdA in self-non-self DNA discrimination.

To further evaluate the role of 6mdA, we transfected 6mdA-DNA into the cells. The qRT-PCR and ELISA analyses indicated that DNA 6mdA enhanced the immune stimulatory potential of incoming DNA (Figures 5F and 5G). DNA 6mdA enhanced the phosphorylation of IFN regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1), suggesting that DNA 6mdA enhances the activity of DNA-sensing pathway (Figure 5H). Moreover, RNA m6A could further elevate the potential of 6mdA to increase immune stimulatory potential of incoming DNA (Figures 5I and 5J). And the potential of 6mdA and m6A to increase the IFNb and cGAMP productions could be abrogated by cGAS KO (Figures 5K–5M) or inhibition (Figures S5H–S5J). These results indicated that RNA m6A and DNA 6mdA medications cooperatively elevated the cGAS activation in the cellular context.

Moreover, we evaluated the role of 6mdA in the intracellular condensation of the incoming DNA. As shown in Figure 5N, 6mdA enhanced the DNA-condensation, while m6A further elevated the potential of 6mdA to increase DNA-condensation. Moreover, among YF1–3, YF2 KD most notably decreased the cooperation between m6A and 6mdA to elevate the DNA-condensation (Figure 5O), in line with the in vitro results. Consistently, YF2 KD reduced the cooperative potential between m6A and 6mdA to stimulate the IFNb production (Figures 5P and 5Q).

Discriminative incorporation of 6mdA into viral DNA elevates the potential of virus to activate host immune surveillance

Viruses of mammals display extremely low 6mdA, while viruses (phages) of bacteria and lower eukaryotes show high 6mdA, even up to 37% (Figure 6A).⁹^,¹⁵^,³⁸ The extremely low 6mdA in the DNAs of mammalian viruses suggests that viral DNA 6mdA modification provides a selective pressure under host immune surveillance, and mammalian viruses might be selected to lose 6mdA to evade the host surveillance. To evaluate this hypothesis, we examined the in vivo effect of 6mdA via increasing the 6mdA levels in mammalian viral DNAs. Considering the general low fidelity of microbial nucleotide polymerases, we speculated that 6mdA could be indiscriminately incorporated into viral DNAs. We first determined whether 6mdA could be incorporated into the DNA by microbial DNA-directed DNA polymerases (rTaq and Q5 PCR polymerases) and RNA-directed DNA polymerase (reverse transcriptase). By replacing dATP with N⁶-methyl-dATP (6mdATP), we observed an efficient incorporation of 6mdA into the DNA products (Figure 6B). The incorporation ratio of 6mdA/dA into DNA was almost equal to the input ratio (Figure S6A). And the 6mdA incorporation increased the immune stimulatory potential of the DNA products (Figure S6B). These results suggested that 6mdA levels in the viral DNAs could be elevated by the supplementation of 6mdA nucleoside.

We then added 6mdA nucleoside into the medium for the propagation of Adv or herpes simplex virus (HSV). Supplementation with 50 μM 6mdA increased the free 6mdA in cytoplasm to more than 20% (Figure 6C) and the 6mdA in the viral DNAs up to 4% (Figure 6D). Notably, 6mdA supplementation poorly increased 6mdA levels in mammalian DNAs only to less than 0.002% (Figure 6E), suggesting that 6mdA supplementation led to a discriminative incorporation of 6mdA into viral DNA. To evaluate the immune stimulatory potential of viral DNA, the cells were infected with 6mdA virus. As shown in Figure 6F, the incorporation of 6mdA into viral DNAs enhanced the immune stimulatory potential of viruses. And cGAS KO abrogated the capacity of 6mdA incorporation to enhance the stimulatory potential of virus (Figures S6C and S6D).

The above results raised a possibility of discriminative incorporation of nucleotide analogue into pathogenic nucleic acids to elevate host immune surveillance (DINA-EHIS) (Figure 6G). Older adults are generally susceptible to virus infection. A central mechanism for this susceptibility is that, in older adults, virus DNA sensing is frequently impaired, and infection might not effectively mount host immune surveillance. In line with the observations in human, HSV infection stimulated much more IFNb production in young mice than in aged mice (Figure S6E). Consistently, young mice could well tolerate the HSV infection in a cGAS-dependent manner,³¹ while aged mice could not (Figure S6F). Notably, 6mdA incorporation elevated the immune stimulatory potential of virus in aged mice (Figures S6E and S6F), suggesting a possibility for DINA-EHIS.

However, 6mdA nucleoside showed poor membrane permeability and inefficient phosphorylation by intracellular kinase(s), which limits the 6mdATP formation in vivo. To bypass these limitations, a ProTide prodrug strategy³⁹ was applied to design compound QKY-613 (Cas number: 2484713-92-8) (Figures S6G and S6H). Based on the ProTide principle,³⁹ phosphorylated 6mdA nucleotide could be released from QKY-613 by intracellular enzymes (Figure 6H). Unlike 6mdA nucleoside, 200 nM QKY-613 could effectively elevate the intracellular free 6mdA level (Figure 6I) to result in effective 6mdA incorporation into viral DNA (Figure 6J). Due to the remarkable fidelity difference between viral and mammalian nucleotide polymerases, 6mdA could not be effectively incorporated into mammalian DNA (Figure 6K). Consistently, ELISA analyses indicated that solo QKY-613 treatment showed little impact on IFNb levels (Figure S6I), suggesting that QKY-613 does not cause the immune surveillance system to misrecognize self-DNA. QKY-613-viruses showed the enhanced stimulatory potential in the MEF cells, which could be abrogated by cGAS KO (Figures 6L, S6J, and S6K). In the context of aged mice infected with HSV, QKY-613 elevated immune responses (Figure 6M) and reduced mortality (Figure 6N). Interestingly, KO and overexpression of FTO increased and decreased IFNb production in response to QKY-613 virus infection (Figures 6O and 6P), respectively. These results suggested a role for the modification eraser(s) in the DINA-EHIS.

Collectively, these results indicated that discriminative incorporation of 6mdA into viral DNA could elevate host immune surveillance.

Discussion

Self-non-self discrimination is central to life,² but the recognition of the incoming DNAs among abundant self-DNAs is a substantial challenge. To recognize non-self-DNA, bacteria developed many modification-based mechanisms such as the restriction-modification system.¹⁵ However, the mammalian cytosolic DNA sensors indiscriminately bind the DNA,⁵^,⁶ suggesting specific mechanisms for self-non-self discrimination. Interestingly, mammals show fewer DNA modifications and more RNA modifications. Here, we show that host RNA and incoming DNA modifications cooperatively elevate the cytosolic condensation potential of incoming DNA to mount immunosurveillance. These results suggest that mammals might be evolved to reduce DNA modification diversity and increase RNA modification diversity to facilitate self-non-self discrimination.

Our results build a direct link between immune surveillance and the diversity imbalance of nucleic acid modifications. There are at least two diversity imbalances: (1) between mammalian and invertebrate DNAs: the genomic DNAs of microbes contain multiple modifications, while mammalian DNAs have only one abundant modification (5mdC)⁹; and (2) between mammalian DNA and RNA: unlike mammalian DNAs, mammalian RNAs show more than 60 modifications.⁴⁰ Interestingly, mammalian RNAs and microbial DNAs share multi modifications,⁴¹ and numerous modified RNA readers interact with the nucleic acid sensor(s) in mammals.²¹^,²²^,⁴²^,⁴³^,⁴⁴ This study suggests that RNA and DNA modifications might cooperatively contribute to self-non-self discrimination. Our results suggest that the diversity imbalances of nucleic acid modifications might provide an evolutionary advantage for mammals. This hypothesis is supported at least partially by the facts that mammalian viruses display extremely low 6mdA, while viruses (phages) of bacteria show high 6mdA.⁹^,¹⁵^,³⁸ The extremely low 6mdA in the DNAs of mammalian viruses suggests that microbial DNA 6mdA provides a selective pressure under host immune surveillance. It will be interesting to determine whether other microbial DNA modifications such as N⁴-methyldeoxycytosine and corresponding mammalian RNA modification-binding proteins are involved in incoming DNA sensing.

This study reveals a role of base modifications and modified base-binding proteins in immune surveillance, which might be targeted for therapeutic interventions. DNA sensing is well documented to control immune surveillance.²^,⁴⁵^,⁴⁶ Consistently, chemotherapies and radiotherapies can modify DNA to control surveillance.⁴⁵ However, cytosolic DNA sensors indiscriminately bind the phosphate backbones of DNA,⁴⁵ necessitating specific mechanism(s) for recognition. Here, we show that base modifications and modified base-binding proteins might control immune recognition via condensation (phase separation), raising a possibility for the development of new therapeutic strategies. This is supported at least partially by the fact that, as classical chemotherapies, alkylating agents are well documented to modify nucleic acids and mount anti-cancer immune surveillance.⁴⁷^,⁴⁸ Additionally, numerous studies have shown that DNA-sensing plays a key role in anti-cancer immune surveillance, the basis of immunotherapy.²^,⁴⁵^,⁴⁶ Our results indicate that modified base-binding proteins can regulate the activity of DNA-sensing pathways. Hence, the levels of modified base-binding proteins might predict the outcome of immunotherapy. It will be interesting to investigate the association between modified base-binding protein levels and immunotherapy outcome.

Our results suggest a possibility of DINA-EHIS for therapeutic intervention (Figure 6Q). Most traditional antiviral strategies are based on separately targeting either the virus or host protein(s).⁴⁹^,⁵⁰ Virus-targeting strategy is likely to yield higher specificity and lower toxicity, but a narrow antiviral activity spectrum and a higher likelihood of resistance developing.⁴⁹^,⁵⁰ Conversely, a host-targeting strategy, such as systemic IFN administration, might lead to a broader antiviral spectrum and less resistance, but higher toxicity particularly in older adults.⁴⁹^,⁵⁰ The DINA-EHIS strategy might target both virus and host. Instead of targeting a specific viral protein, DINA-EHIS targets the low fidelity of viral nucleotide polymerase, a universal characteristic of all microbes. To target the host, DINA-EHIS might elevate self-non-self discrimination instead of systemically boosting the immune responses. Hence, DINA-EHIS might lead to a broader spectrum, higher specificity, and lower toxicity. And the clinical potential of this strategy is supported at least by the facts that, in older adults, virus infection less effectively mounts host immune surveillance. Notably, like virus, cancer cells usually apply nucleotide polymerase with low fidelity to synthesize their nucleic acids, which plays a key role in the high mutation and drug resistance.⁵¹^,⁵² This might enable DINA-EHIS to function as a potential strategy to treat cancer. Based on the DINA-EHIS strategy, the discriminative incorporation of nucleotide analogue into the nucleic acids of cancer cells might boost immune system to recognize and subsequently kill cancer cells. Additionally, we noted that the erasers of nucleic acid modification such as FTO might influence the outcome of DINA-EHIS. These observations further support our results that, although sensors bind the phosphate backbones but not the bases of DNA, the base modifications can still regulate immune surveillance. It has been well documented that the expression of modification erasers might predict the outcome of alkylating agents to modify nucleic acids to kill cancer cells.⁵³ Accordingly, we propose that the expression of modification erasers might also predict the outcome of DINA-EHIS.

However, several limitations of this study should be noted. First, although host RNA and incoming DNA modifications increase the immune stimulatory potential of DNA dominantly via cGAS, we did not exclude the potential role(s) of other DNA sensor(s). Due to the crucial role of incoming DNA condensation in the activation of cytosolic DNA sensors,⁸^,⁵⁴ we speculate that RNA and incoming DNA modifications likely contribute to the activation of other DNA sensors. Second, as m6A- and/or 6mdA-binding proteins, only YFs were studied in this study. However, there are numerous m6A- and/or 6mdA-binding proteins in mammalian cells. Consistently, YF knockdown reduced but not abrogated the incoming DNA condensation. Due to the number of these proteins,⁴⁴^,⁵⁵ more work is needed to determine the role of these modified base-binding proteins. Third, our study focused on the immediate immune response to the incoming DNA. RNA and DNA modifications play roles in degradation, and DNA and/or RNA degradation might reduce the response in a time-dependent manner. Hence, it will be interesting to determine the time course effects of RNA and/or DNA modifications. Despite these limitations, our study still reveals a cooperative role of RNA and DNA modifications in immune surveillance and a possibility of DINA-EHIS.

Materials and methods

Cell culture and transfection

HEK293, RAW264.7, Vero, HEK293T, and HeLa cells were maintained in DMEM containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and streptomycin at 37°C under a humidified atmosphere of 5% CO₂. C2C12 and THP-1 cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10% FBS. MEF and MLF cells were prepared and maintained as previously described.³¹^,⁵⁶ According to the manufacturer’s instructions, DNA and Dam protein transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific Inc.) and jetCRISPR (Polyplus-transfection, Illkirch, France), respectively.

Reagents

Anti-Flag (Cat. number: 2368), anti-ACTIN (Cat. number: 4967), anti-IRF3 (Cat. number: 4947), anti-phospho-IRF3 (Ser396) (Cat. number: 83611), anti-mouse cGAS (Cat. number: 31659), anti-TBK1 (Cat. number: 3504) and anti-phospho-TBK1 (Ser172) (Cat. number: 5483) antibodies were from Cell Signaling (Beverly, MA, USA). Anti-FTO (Cat. number: ab92821) and anti-HA (Cat. number: ab9110) antibodies were from Abcam (Cambridge, UK). Anti-YTHDF1 (Cat. number: 17479-1-AP), anti-YTHDF2 (Cat. number: 24744-1-AP), anti-YTHDF3 (Cat. number: 25537-1-AP), and anti-G3BP1 mouse (Cat. number: 66486-1-lg) antibodies were from Proteintech (Wuhan, Hubei, China). Anti-N⁶-methyladenosine antibody (Cat. number: 202003) was from Synaptic Systems (Göttingen, Germany). Replication-defective Adv Ad5-GFP vectors (AdV-GFP) deleted in E1 and E3 coding domains was from Microbix Biosystems (Mississauga, Canada). HSV-1-GFP was a generous gift from Professor Erguang Li at the Nanjing University in China.

Full-length human cGAS (GenBank: NM_138441.3) sequence was cloned into the Nco I and Sal I sites of pET28a vector (Novogen Limited, Hornsby Westfield, NSW, Australia) to build pET-28a-cGAS plasmid for the expression of cGAS protein. Full length cGAS was cloned into the Not I and Xba I sites of pCDNA3.1 vector to build pCDNA3.1 HA-cGAS plasmid. Full-length E. coli Alkb (GenBank: NP_416716.1), Dam (GenBank: NP_417846.1) and human YTHDF1–3 (GenBank: NM_017798.4, NM_016258.3, NM_001277813.2) sequences were cloned into the EcoR I and Xho I sites of pET28a vector to build the plasmids for the in vitro expression. Full-length human YBX3 (GenBank: NM_003651.5) sequence was cloned into the EcoR I and Hind III sites of pET28a vector to build the plasmid. Full-length human CAPRIN1 (GenBank: NM_005898.5) sequence was cloned into the Sac I and Xho I sites of pET28a vector to build the plasmid. Full-length human IGF2BP1 (GenBank: NM_006546.4), ILF3 (GenBank: NM_001394824.1) and CPSF7 (GenBank: NM_024811.4) sequences were cloned into the EcoR I and Not I sites of pET28a vector to build the plasmids for the in vitro expression. Full-length YTHDF2 was also cloned into the EcoR I and Xho I sites of pCDNA3.1 vector to build pCDNA3.1 Flag-YTHDF2-FL plasmid. YTHDF2 amino acid 1–384 and 385–579 sequences were cloned into the EcoR I and Xho I sites of pCDNA3.1 vector to build pCDNA3.1 Flag-YTHDF2-N and pCDNA3.1 Flag-YTHDF2-C-truncated mutant plasmids, respectively. Full-length FTO (GenBank: NM_011936.2) was also cloned into the EcoR I and Not I sites of pCDNA3.1 to build pCDNA3.1 FTO plasmid.

N⁶-methyl-2′-deoxyadenosine (6mdA) (Cat. number: M2389), 2′-deoxyadenosine (dA) (Cat. number: D7400), and RU.521 (Cat. number: SML2347) were from Sigma-Aldrich (St. Louis, MS, USA). UZH1a (Cat. number: HY-134673A), adenosine (A) (Cat. number: HY-B0228) and N⁶-methyladenosine (m6A) (Cat. number: HY-N0086) were from MedChemExpress (Shanghai, Shanghai, China). N⁶-methyl-2′-deoxyadenosine-5′-triphosphate (6mdATP) (Cat. number: Axbio-an) and 2′3′-cGAMP (Cat. number: tlrl-nacga23) was from Axbio (Shenzhen, Guangzhou, China) and InvivoGen (San Diego, CA, USA), respectively. ATP (Cat. number: R0441), GTP (Cat. number: R046), and streptavidin dynabeads (Cat. number: 65607D) were from Thermo Fisher Scientific. Small interfering RNA (siRNA)-dependent knockdown was carried out following the manufacturer’s protocol (Dharmacon, Lafayette, CO, USA). Non-targeting siRNA from Dharmacon was used as the control.

Visualization of cGAS-DNA complex structure

cGAS-DNA complex structure (PDB: 5N6I) was derived from the PDB database (https://www.rcsb.org). The cGAS-DNA complex model was visualized by PyMOL (version 2.5.2) using default parameters.

qRT-PCR

qRT-PCR was performed as described in our previous study.⁵⁷ Briefly, RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription was carried out using the PrimeScript RT Reagent Kit (TaKaRa, DaLian, Liaoning, China). qRT-PCR was performed using the SYBR PrimeScript PCR Kit II (TaKaRa). The primers used for qRT-PCR are described in Table S2.

Immunoblotting analyses

Immunoblotting analyses were performed as previously described.⁵⁷ The cells were harvested in the RIPA lysis buffer containing 1× PBS, 0.1 mg/mL phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, protease inhibitor cocktail (Roche, Indianapolis, IN, USA), and phosphatase inhibitor cocktail (Roche). The cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and blotted with the noted antibodies.

IF analyses

IF analyses were carried out as previously described.⁵⁷ The cells were plated on the coverslips precoated with type I collagen. After the noted treatments, the cells were fixed with 4% formaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. The resultant cells were incubated with rabbit (or mouse) primary antibody for 1.5 h, followed by incubation with the fluorescence-labeled goat anti-rabbit (or anti-mouse) immunoglobulin G (IgG) (Thermo Fisher Scientific Inc.) for 1 h. Then, the cells were incubated with mouse (or rabbit) primary antibody for 1.5 h. After incubation with the fluorescence-labeled goat anti-mouse (or anti-rabbit) IgG for 1 h, the coverslip was mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA).

Immunoprecipitation

Immunoprecipitation assays were performed as described in our previous study.⁵⁸ Cells were harvested in E1A lysis buffer (0.1% NP40, 5 mM EDTA, 250 mM NaCl, 50 mM HEPES [pH 7.5], protease inhibitor cocktail [Roche], phosphatase inhibitor cocktail [Roche]). The resultant lysates were incubated with the precipitating antibody for 6 h, followed by incubation with protein G beads (Invitrogen, Carlsbad, CA, USA) at 4°C for overnight. After washing, the precipitates were denatured in SDS protein sample buffer at 95°C and resolved by SDS-polyacrylamide gel electrophoresis.

ELISA assays

Briefly, ELISAs were performed using ELISA kits from BioLegend (San Diego, CA, USA) as described in our previous study.⁵⁷ The sera or cell culture supernatants were mixed with 50 μL of Assay Buffer A for 2 h. Then, 100 μL of the appropriate antibody solution was added, followed by incubation at room temperature for 2 h. After washing, 100 μL avidin-HRP D solution was added and incubated for 30 min. After washing, 100 μL Substrate Solution F was added, followed by incubation for 30 min in the dark. The reactions were stopped by 100 μL Stop Solution. We measured 450 nm absorbance of each sample, and the IFNb protein levels were quantified according to the standard curves.

Quantification of A and m6A by liquid chromatography-mass spectrometry/mass spectrometry

MS analyses of RNA A and m6A were performed as previously described.²⁸ Briefly, RNA was digested by nuclease P1 (Sigma) at 37°C for 12 h, followed by incubating with alkaline phosphatase at 37°C for 2 h. RNA solution was diluted 10 times, and the 10 μL solution was injected into liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The nucleosides were separated by reverse phase high-performance liquid chromatography on an Agilent C 18 column, followed by MS detection using a Qtrap5500 triple quadrupole mass spectrometer (AB SCIEX, Concord, Ontario, Canada) in positive ionization mode. Multiple-reaction-monitoring (MRM) mode was applied to monitor A and m6A parent ion to product ions: m/z 267.9 to 136.1 for A (collision energy [CE]: 25 V; declustering potential [DP]: 10 V); m/z 282.1 to 150.1 for m6A (CE 30 V; DP 12 V). The mobile phases (delivered at 0.40 mL/min) consisted of H₂O for mobile phase A and methanol for mobile phase B. An isocratic elution (72% mobile phase A and 28% mobile phase B) was performed at stop time of 3 min. The Analyst software for Windows (Version, 1.6.1) was used for peak area quantification and data processing. The A and m6A levels were quantified according to the authentic standard curves.

Quantification of cGAMP by LC-MS/MS

Quantification of cGAMP by LC-MS/MS was performed as described in our previous study.⁵⁷ The samples were separated using the ACQUITY UPLC system (Waters), followed by MS detection using a Qtrap5500 triple quadrupole mass spectrometer (AB SCIEX) in positive electrospray ionization mode. MRM mode was applied to monitor cGAMP parent ion to product ions: m/z 675 to 136 (CE, 45 V; DP, 70 V). The mobile phases (delivered at 0.45 mL/min) consisted of H₂O (containing 20 mM NH₄Ac and 0.05% HAc) for A and methanol (containing 20 mM NH₄Ac and 0.05% HAc) for B. An isocratic elution (50% A and 50% B) was performed for 5 min. The Analyst software for Windows (Version, 1.6.1) was used for peak area quantification and data processing. The cGAMP levels were quantified according to the authentic standard curve.

Meta-analyses

M6A binding,⁴⁴^,⁵⁵ cGAS binding,²¹ and SG proteins³³^,⁵⁹ from meta-analyses were described in Table S1. Prediction of protein phase separation was performed as previous described.³³

Expression and purification of proteins

Briefly, E. coli strain BL21 (DE3) was separately transformed with plasmids encoding the proteins. The proteins were expressed and purified as described in our previous studies.²⁸^,⁵⁷ Concentration of resultant proteins and buffer exchange were performed using an Amicon Ultrafree centrifugal filter (Millipore, Burlington, MA, USA) with a cutoff of 10 kD. The proteins were labeled with Alexa Fluor 568 using the Alexa Fluor 568 Protein Labeling Kit (Thermo Fisher Scientific, Inc.).

In vitro condensation (phase separation) assay

In vitro cGAS-DNA condensation (phase separation) analyses were performed as previously described.⁵⁷ Protein was mixed with DNA and or RNA in a buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The mixtures were incubated at 37°C for the noted time. The turbidity was measured at 340 nm using a NanoDrop 2000C (Thermo Fisher Scientific, Inc.). Fluorescence images of the mixtures were captured by fluorescence microscopy (Olympus IX81, Tokyo, Japan). To determine condensate diagram, fluorescent microscope with a 100× oil objective was used as previously described.⁵⁷ Condensate formation was determined by the detection of at least one visible condensate in three random fields. All analyses were performed in triplicate.

MARTFQ assay

MARTFQ assay was performed as described in our previous study.²⁸ Briefly, 50 nM of noted protein was mixed with a serial dilution of DNA as noted. After incubation for 10 min, the samples were subjected to fluorescence analyses based on 348-nm intrinsic protein fluorescence excited by 280 nm light. The binding constant was estimated according to the modified Stern-Volmer equation: RF0/ΔRF = (1/fK) × (1/[Q]) + (1/f), where ΔRF is equal to RF0 (the protein fluorescence intensity in the absence of metabolite) − RF (the intensity in the presence of metabolite); f is the fractional maximum fluorescence intensity of protein; K is the quenching constant, considered as the binding constant; and [Q] is the concentration of metabolite.

EMSA

EMSA was carried out as described in our previous study.⁵⁷ Briefly, 50 nM Cy3-labeled DNA was mixed with a serial dilution of the indicated proteins in a buffer containing 150 mM NaCl and 20 mM Tris-HCl (pH 7.5). After incubation at 37°C for 10 min, the mixtures were analyzed on 1.5% agarose gel. Images were acquired by ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and analyzed by ImageJ.

MST assay

MST assay was performed as described in our previous study.⁵⁷ Briefly, 50 nM Cy5-labeled DNA was mixed with a serial dilution of the indicated proteins in a buffer containing 150 mM NaCl and 20 mM Tris-HCl (pH 7.5). Then, the thermophoretic movement of DNA with protein was analyzed by Monollith NT.115 instrument (Nanotemper Technologies, Munich, Germany).

cGAS activity assay

In the presence or absence of RNA and YTHDFs, purified cGAS protein at the noted concentrations was mixed with DNA in the buffer containing 5 mM MgCl₂, 20 mM Tris-HCl (pH 7.5), 2 mM ATP, and 2 mM GTP. After incubation at 37°C for 2 h, the mixtures were diluted and centrifuged at 16,000×g for 10 min. The resultant supernatants were analyzed by MS as described above.

Incorporation of 6mdA into DNA by PCR DNA polymerase

PCR reactions were performed using rTaq DNA polymerase (Cat. number: TAP-201, Takara, Beijing, China) or Q5 high-fidelity DNA polymerase (Cat. number: M0491L, New England BioLabs, Ipswich, MA, USA). We used 1 ng EGFP DNA fragment as the template, and mixed with 0.2 μM primer(s), 1 U DNA polymerase, dCTP, dTTP, dGTP, and dATP (or 6mdATP) as noted in a total volume of 50 μL. The PCR products were analyzed by 1.5% agarose gel electrophoresis and were extracted using a PCR clean up kit (QIAGEN). The resultant extracted DNA was analyzed by MS or dot blotting as described in our previous study.⁵⁸

Incorporation of 6mdA into cDNA by RNA-directed DNA polymerase (reverse transcriptase enzyme)

Total RNA from MEF cells was isolated as described in our previous study.²⁸ cDNA synthesis was performed with RevertAid M-MuLV RT (Cat. number: EP0441, Thermo Fisher Scientific Inc.), an RNA-directed DNA polymerase (reverse transcriptase enzyme). Five micrograms RNA was mixed with 1 μM Oligo (dT)₁₈ primer, 4 μL of 5× reaction buffer, 10 U RevertAid M-MuLV RT, dCTP, dTTP, dGTP, and dATP or 6mdATP in a total volume of 20 μL. Then, cDNA products were purified using QIAquick Nucleotide Removal Kit (QIAGEN) to remove free dNTP.

Dot blotting analyses of 6mdA

Dot blotting analyses of 6mdA were performed as described in our previous studies.²⁸^,⁵⁸ DNA samples were subjected to extensive RNase treatments before loading on the membrane.

Quantification of dC, 6mdA, and dA by LC-MS/MS

Cellular and viral DNAs were isolated using the cell culture DNA extraction Kit (QIAGEN) and NucleoSpin Virus (Macherey-Nagel, Düren, Germany), respectively. Resultant DNAs were digested by nuclease P and dephosphorylated by alkaline phosphatase. The nucleosides were separated using reverse phase high-performance liquid chromatography with a Waters XBridge C18 column, followed by MS detection using AB SCIEX QTRAP 5500 LC-MS/MS in positive electrospray ionization mode. MRM mode was used to monitor parent ion to product ions: m/z 228 to 112 (CE 15 V; DP 60 V) for dC, m/z 266 to 150 for 6mdA (CE 26 V; DP 100 V), and m/z 252 to 136 for dA (CE 45 V; DP 140 V). The mobile phases (delivered at 0.40 mL/min) consisted of H₂O for A and methanol (containing 0.05% formic acid) for B. An isocratic elution (85% A and 15% B) was performed at a stop time of 5 min. The Analyst software for Windows (Version, 1.6.1) was used for peak area quantification and data processing. The dC, dA and 6mdA levels were quantified according to the authentic standard curves. Viral DNAs were normalized using qRT-PCR with the primers described in Table S2. dA levels in PCR products were normalized by dC in the PCR products.

Propagation of HSV and Adv

HSV-1-GFP and Adv-GFP viruses were propagated in Vero and HEK293 cells, respectively. All viruses were tittered in Vero cells. For the generation of 6mdA-incorperated viruses, the cells were infected with HSV-1-GFP or Adv-GFP in serum-free medium. After 2 h, the virus-containing medium was replaced with the medium containing 10% FBS and the noted compound at the indicated concentration until viruses were harvested.

Synthesis of QKY-613

Preparation of Compound 1

Under an argon atmosphere, L-alanine benzyl ester hydrochloride (21.568 g, 0.1 mol, 1 eq) in methylene chloride (200 mL) was added into triethylamine (21.25 g, 0.21 mol, 2.1 eq), followed by cooling to −78°C. Phenyl dichlorophosphate (23.2 g, 0.11 mol, 1.1 eq) was then added dropwise, followed by stirring at −78°C for 30 min. Then, the mixture was stirred at room temperature for 3 h. After being cooled to 0°C, 4-nitrophenol (13.91 g, 0.1 mol, 1 eq) and triethylamine (10.12 g, 0.1 mol, 1 eq) were added. After being stirred at 0°C for 30 min and subsequently at room temperature for 5 h, the solvent was removed in situ and the product was purified by column chromatography (SiO₂; PE: EA = 3: 1) as an oil: 23 g, yield 50.4%. ¹H NMR (400 MHz, CDCI3): δ 8.11–8.18 (d, 2H), 7.18–7.36 (m, 12H), 5.08–5.16 (m, 2H), 4.13–4.34 (m, 2H), 1.39–1.42 (d, 3H); MS (ESI) m/z: 457.1 (M + H)⁺.

Preparation of QKY-613

Under an argon atmosphere, 6mdA (5 g, 18.85 mmol, 1eq) was dissolved in 120 mL anhydrous tetrahydrofuran (THF) and 40 mL N-methylpyrrolidone (NMP). Then, 37.7 mL Tert-butyl magnesium chloride (1 M in THF, 37.7 mmol, 2 eq) was added dropwise, followed by stirring for 20 min. Compound 1 (17.2 g, 37.7 mmol, 2 eq) in 50 mL anhydrous THF was added slowly. The mixture was heated to 55°C for 7 h, followed by cooling to room temperature. The mixture was poured into a 10% aqueous solution of NH₄Cl (500 mL) and extracted with dichloromethane (DCM). The combined organic layer was dried over Na₂SO₄, filtered and evaporated under reduced pressure. The residue was purified by flash column chromatography on silica gel using DCM: MeOH = 95: 5 as elution to give the title compound as a white solid, 0.67 g, yield 6.1%. 1H NMR (400 MHz, DMSO-d6): δ 8.29 (d, 1H), 8.24 (s, 1H), 7.76 (s, 1H), 7.29–7.33 (m, 7H), 7.15–7.19 (m, 3H), 6.39 (m, 1H), 6.11 (m, 1H), 5.62 (m, 1H), 5.06 (m, 2H), 4.47 (m, 1H), 4.24 (m, 1H), 4.04–4.13 (m, 2H), 3.89 (m, 1H), 2.95 (s, 3H), 2.74 (m, 1H), 2.33 (m, 1H), 1.22 (m, 3H); MS (ESI) m/z: 583.3 (M + H)⁺.

Mice

The animal handling procedures were approved by the Animal Care and Use Committee of DaLian Medical University (Approval number: AEE22079). FTO KO mouse model (C57BL/6) was generated as described in our previous study.²⁸ cGAS KO mouse model (C57BL/6) was generated as described in our previous study.⁵⁷ Briefly, we designed a construct resulting in the deletion of 2–4 exons of the cGAS coding sequence. Cas9 mRNA and sgRNA generated by in vitro transcription were then injected into fertilized eggs for KO mouse productions. The founders were genotyped by PCR (F1: 5′-TATGTACAGGAACCCGTGCAG-3’; R1: 5′-CTTAACCACTGAGCCATCTCTAG-3′) followed by DNA sequencing analysis. The positive founders were bred to the next generation (F1) and subsequently genotyped by PCR (F2: 5′-TTCACTAAATAGACCAAGCTGCTG-3’; R2: 5′-ATGACTCAGCGGATTTCCTCG-3′), DNA sequencing and immunoblotting analyses. All mice were housed in a specific pathogen free facility at 22 ± 2°C under a cycle of 12 h light (7:00 am light on) and 12 h dark (7:00 pm light off).

Intravenous infection with HSV 1 was performed as previously described.³¹ For the measurement of IFNb production, the mice were infected intravenously with the noted virus at 1 × 10⁷ plaque-forming units (pfu) per mouse and then sera were collected to measure the IFNb levels. For survival experiment, the mice were infected intravenously with 1 × 10⁶ pfu HSV per mouse, and mouse survival was monitored for 8 days. For QKY-613 supplementation, mice were treated intraperitoneally with 10 mg/kg QKY-613 once a day.

Statistical analyses

All statistical tests, comparisons, replications, and sample sizes are included in the figure legends. All data are shown as mean ± standard deviation. Error bars indicate standard deviation. For Kaplan-Meier analyses, log rank test was used to calculate the p values. All other statistical analyses were performed using unpaired two-tailed Student’s t-test. In all cases, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, N.S. = not statistically significant (p > 0.05). The experiments were replicated at least three times, and all attempts at replication were successful. All statistical analyses were performed using Prism 9 (GraphPad Software, GraphPad Prism, San Diego, CA, USA).

Data and code availability

All data that support the findings of this study are available with the paper and its supplemental information. All unique reagents generated in this study will be made available upon request. An agreement with our institute’s Materials Transfer Agreement (MTA) may be required.

Acknowledgments

We sincerely appreciate Professor Zhijian J. Chen at the University of Texas Southwestern Medical Center for providing the information for the purification and condensation of cGAS. We gratefully acknowledge the services provided by the Technology Core Facility at DaLian Medical University. This study was supported by the grant from Open Fund of Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu University), Ministry of Education of China (NO. KF2023009 to L.W.), and Science and Technology Innovation Team Project of Basic Scientific Research Project of Liaoning Provincial Department of Education (L.W.).

Author contributions

L.W. and Q.Y. conceived the project and designed most experiments. C.S. wrote the manuscript. N.W. and Q.L. performed most experiments. B.W., Z.Y., and C.S. carried out bioinformatics analyses. Q.L., Y.H., R.L., and X.L. expressed and purified the proteins. S.L., Z.Y., J.F., and S.W. maintained cells and raised the mice. Z.S., H.W., and L.D. constructed the plasmids. Y.W. and C.Q. provided reagents.

Declaration of interests

The authors declare that no competing interests exist.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.10.027.

Contributor Information

Qingkai Yang, Email: yangqingkai@dmu.edu.cn.

Lina Wang, Email: li-nawang@dmu.edu.cn.

Supplemental information

Document S1. Figures S1–S6 and Table S2

mmc1.pdf^{(2.1MB, pdf)}

Table S1. The candidate genes from meta analyses

mmc2.xlsx^{(30.1KB, xlsx)}

Document S2. Article plus supplemental information

mmc3.pdf^{(6.8MB, pdf)}

References

1.Schlee M., Hartmann G. Discriminating self from non-self in nucleic acid sensing. Nat. Rev. Immunol. 2016;16:566–580. doi: 10.1038/nri.2016.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Bartok E., Hartmann G. Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids. Immunity. 2020;53:54–77. doi: 10.1016/j.immuni.2020.06.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Sun L., Wu J., Du F., Chen X., Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013;339:786–791. doi: 10.1126/science.1232458. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Wu J., Sun L., Chen X., Du F., Shi H., Chen C., Chen Z.J. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 2013;339:826–830. doi: 10.1126/science.1229963. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Li X., Shu C., Yi G., Chaton C.T., Shelton C.L., Diao J., Zuo X., Kao C.C., Herr A.B., Li P. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity. 2013;39:1019–1031. doi: 10.1016/j.immuni.2013.10.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Andreeva L., Hiller B., Kostrewa D., Lässig C., de Oliveira Mann C.C., Jan Drexler D., Maiser A., Gaidt M., Leonhardt H., Hornung V., Hopfner K.P. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders. Nature. 2017;549:394–398. doi: 10.1038/nature23890. [DOI] [PubMed] [Google Scholar]
7.Zhou W., Whiteley A.T., de Oliveira Mann C.C., Morehouse B.R., Nowak R.P., Fischer E.S., Gray N.S., Mekalanos J.J., Kranzusch P.J. Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance. Cell. 2018;174:300–311.e11. doi: 10.1016/j.cell.2018.06.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Du M., Chen Z.J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science. 2018;361:704–709. doi: 10.1126/science.aat1022. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Wion D., Casadesús J. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat. Rev. Microbiol. 2006;4:183–192. doi: 10.1038/nrmicro1350. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Schiffers S., Ebert C., Rahimoff R., Kosmatchev O., Steinbacher J., Bohne A.V., Spada F., Michalakis S., Nickelsen J., Müller M., Carell T. Quantitative LC-MS Provides No Evidence for m(6) dA or m(4) dC in the Genome of Mouse Embryonic Stem Cells and Tissues. Angew. Chem. Int. Ed. Engl. 2017;56:11268–11271. doi: 10.1002/anie.201700424. [DOI] [PubMed] [Google Scholar]
11.O'Brown Z.K., Boulias K., Wang J., Wang S.Y., O'Brown N.M., Hao Z., Shibuya H., Fady P.E., Shi Y., He C., et al. Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA. BMC Genom. 2019;20:445. doi: 10.1186/s12864-019-5754-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Fu Y., Luo G.Z., Chen K., Deng X., Yu M., Han D., Hao Z., Liu J., Lu X., Dore L.C., et al. N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell. 2015;161:879–892. doi: 10.1016/j.cell.2015.04.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Greer E.L., Blanco M.A., Gu L., Sendinc E., Liu J., Aristizábal-Corrales D., Hsu C.H., Aravind L., He C., Shi Y. DNA Methylation on N6-Adenine in C. elegans. Cell. 2015;161:868–878. doi: 10.1016/j.cell.2015.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Zhang G., Huang H., Liu D., Cheng Y., Liu X., Zhang W., Yin R., Zhang D., Zhang P., Liu J., et al. N6-methyladenine DNA modification in Drosophila. Cell. 2015;161:893–906. doi: 10.1016/j.cell.2015.04.018. [DOI] [PubMed] [Google Scholar]
15.Krüger D.H., Bickle T.A. Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts. Microbiol. Rev. 1983;47:345–360. doi: 10.1128/mr.47.3.345-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Dominissini D., Moshitch-Moshkovitz S., Schwartz S., Salmon-Divon M., Ungar L., Osenberg S., Cesarkas K., Jacob-Hirsch J., Amariglio N., Kupiec M., et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485:201–206. doi: 10.1038/nature11112. [DOI] [PubMed] [Google Scholar]
17.Gao Y., Pei G., Li D., Li R., Shao Y., Zhang Q.C., Li P. Multivalent m(6)A motifs promote phase separation of YTHDF proteins. Cell Res. 2019;29:767–769. doi: 10.1038/s41422-019-0210-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Ries R.J., Zaccara S., Klein P., Olarerin-George A., Namkoong S., Pickering B.F., Patil D.P., Kwak H., Lee J.H., Jaffrey S.R. m(6)A enhances the phase separation potential of mRNA. Nature. 2019;571:424–428. doi: 10.1038/s41586-019-1374-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Cheng Y., Xie W., Pickering B.F., Chu K.L., Savino A.M., Yang X., Luo H., Nguyen D.T., Mo S., Barin E., et al. N(6)-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation. Cancer Cell. 2021;39:958–972.e8. doi: 10.1016/j.ccell.2021.04.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Lee J.H., Wang R., Xiong F., Krakowiak J., Liao Z., Nguyen P.T., Moroz-Omori E.V., Shao J., Zhu X., Bolt M.J., et al. Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation. Mol. Cell. 2021;81:3368–3385.e9. doi: 10.1016/j.molcel.2021.07.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Hu S., Sun H., Yin L., Li J., Mei S., Xu F., Wu C., Liu X., Zhao F., Zhang D., et al. PKR-dependent cytosolic cGAS foci are necessary for intracellular DNA sensing. Sci. Signal. 2019;12 doi: 10.1126/scisignal.aav7934. [DOI] [PubMed] [Google Scholar]
22.Liu Z.S., Cai H., Xue W., Wang M., Xia T., Li W.J., Xing J.Q., Zhao M., Huang Y.J., Chen S., et al. G3BP1 promotes DNA binding and activation of cGAS. Nat. Immunol. 2019;20:18–28. doi: 10.1038/s41590-018-0262-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Su R., Dong L., Li Y., Gao M., Han L., Wunderlich M., Deng X., Li H., Huang Y., Gao L., et al. Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion. Cancer Cell. 2020;38:79–96.e11. doi: 10.1016/j.ccell.2020.04.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Abakir A., Giles T.C., Cristini A., Foster J.M., Dai N., Starczak M., Rubio-Roldan A., Li M., Eleftheriou M., Crutchley J., et al. N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat. Genet. 2020;52:48–55. doi: 10.1038/s41588-019-0549-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Zhang C., Chen L., Peng D., Jiang A., He Y., Zeng Y., Xie C., Zhou H., Luo X., Liu H., et al. METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation. Mol. Cell. 2020;79:425–442. doi: 10.1016/j.molcel.2020.06.017. [DOI] [PubMed] [Google Scholar]
26.Lauring A.S., Frydman J., Andino R. The role of mutational robustness in RNA virus evolution. Nat. Rev. Microbiol. 2013;11:327–336. doi: 10.1038/nrmicro3003. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Warren T.K., Jordan R., Lo M.K., Ray A.S., Mackman R.L., Soloveva V., Siegel D., Perron M., Bannister R., Hui H.C., et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016;531:381–385. doi: 10.1038/nature17180. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Wang L., Song C., Wang N., Li S., Liu Q., Sun Z., Wang K., Yu S.C., Yang Q. NADP modulates RNA m(6)A methylation and adipogenesis via enhancing FTO activity. Nat. Chem. Biol. 2020;16:1394–1402. doi: 10.1038/s41589-020-0601-2. [DOI] [PubMed] [Google Scholar]
29.Jia G., Fu Y., Zhao X., Dai Q., Zheng G., Yang Y., Yi C., Lindahl T., Pan T., Yang Y.G., He C. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 2011;7:885–887. doi: 10.1038/nchembio.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Yankova E., Blackaby W., Albertella M., Rak J., De Braekeleer E., Tsagkogeorga G., Pilka E.S., Aspris D., Leggate D., Hendrick A.G., et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021;593:597–601. doi: 10.1038/s41586-021-03536-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Li X.D., Wu J., Gao D., Wang H., Sun L., Chen Z.J. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science. 2013;341:1390–1394. doi: 10.1126/science.1244040. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Vincent J., Adura C., Gao P., Luz A., Lama L., Asano Y., Okamoto R., Imaeda T., Aida J., Rothamel K., et al. Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice. Nat. Commun. 2017;8:750. doi: 10.1038/s41467-017-00833-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Youn J.Y., Dyakov B.J.A., Zhang J., Knight J.D.R., Vernon R.M., Forman-Kay J.D., Gingras A.C. Properties of Stress Granule and P-Body Proteomes. Mol. Cell. 2019;76:286–294. doi: 10.1016/j.molcel.2019.09.014. [DOI] [PubMed] [Google Scholar]
34.Gilbert W.V., Bell T.A., Schaening C. Messenger RNA modifications: Form, distribution, and function. Science. 2016;352:1408–1412. doi: 10.1126/science.aad8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Luecke S., Holleufer A., Christensen M.H., Jønsson K.L., Boni G.A., Sørensen L.K., Johannsen M., Jakobsen M.R., Hartmann R., Paludan S.R. cGAS is activated by DNA in a length-dependent manner. EMBO Rep. 2017;18:1707–1715. doi: 10.15252/embr.201744017. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Li D., Delaney J.C., Page C.M., Yang X., Chen A.S., Wong C., Drennan C.L., Essigmann J.M. Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB. J. Am. Chem. Soc. 2012;134:8896–8901. doi: 10.1021/ja3010094. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Aughey G.N., Cheetham S.W., Southall T.D. DamID as a versatile tool for understanding gene regulation. Development. 2019;146 doi: 10.1242/dev.173666. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Zhang Y., Nelson M., Nietfeldt J., Xia Y., Burbank D., Ropp S., Van Etten J.L. Chlorella virus NY-2A encodes at least 12 DNA endonuclease/methyltransferase genes. Virology. 1998;240:366–375. doi: 10.1006/viro.1997.8936. [DOI] [PubMed] [Google Scholar]
39.Mehellou Y., Rattan H.S., Balzarini J. The ProTide Prodrug Technology: From the Concept to the Clinic. J. Med. Chem. 2018;61:2211–2226. doi: 10.1021/acs.jmedchem.7b00734. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Frye M., Harada B.T., Behm M., He C. RNA modifications modulate gene expression during development. Science. 2018;361:1346–1349. doi: 10.1126/science.aau1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Blow M.J., Clark T.A., Daum C.G., Deutschbauer A.M., Fomenkov A., Fries R., Froula J., Kang D.D., Malmstrom R.R., Morgan R.D., et al. The Epigenomic Landscape of Prokaryotes. Plos Genet. 2016;12 doi: 10.1371/journal.pgen.1005854. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Wang L., Wen M., Cao X. Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science. 2019;365 doi: 10.1126/science.aav0758. [DOI] [PubMed] [Google Scholar]
43.Alarcón C.R., Goodarzi H., Lee H., Liu X., Tavazoie S., Tavazoie S.F. HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell. 2015;162:1299–1308. doi: 10.1016/j.cell.2015.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Arguello A.E., DeLiberto A.N., Kleiner R.E. RNA Chemical Proteomics Reveals the N(6)-Methyladenosine (m(6)A)-Regulated Protein-RNA Interactome. J. Am. Chem. Soc. 2017;139:17249–17252. doi: 10.1021/jacs.7b09213. [DOI] [PubMed] [Google Scholar]
45.Ablasser A., Chen Z.J. cGAS in action: Expanding roles in immunity and inflammation. Science. 2019;363 doi: 10.1126/science.aat8657. [DOI] [PubMed] [Google Scholar]
46.Roers A., Hiller B., Hornung V. Recognition of Endogenous Nucleic Acids by the Innate Immune System. Immunity. 2016;44:739–754. doi: 10.1016/j.immuni.2016.04.002. [DOI] [PubMed] [Google Scholar]
47.Petroni G., Buqué A., Zitvogel L., Kroemer G., Galluzzi L. Immunomodulation by targeted anticancer agents. Cancer Cell. 2021;39:310–345. doi: 10.1016/j.ccell.2020.11.009. [DOI] [PubMed] [Google Scholar]
48.Kwon J., Bakhoum S.F. The Cytosolic DNA-Sensing cGAS-STING Pathway in Cancer. Cancer Discov. 2020;10:26–39. doi: 10.1158/2159-8290.CD-19-0761. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.De Clercq E. Strategies in the design of antiviral drugs. Nat. Rev. Drug Discov. 2002;1:13–25. doi: 10.1038/nrd703. [DOI] [PubMed] [Google Scholar]
50.Bekerman E., Einav S. Infectious disease. Combating emerging viral threats. Science. 2015;348:282–283. doi: 10.1126/science.aaa3778. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Noronha A., Belugali Nataraj N., Lee J.S., Zhitomirsky B., Oren Y., Oster S., Lindzen M., Mukherjee S., Will R., Ghosh S., et al. AXL and Error-Prone DNA Replication Confer Drug Resistance and Offer Strategies to Treat EGFR-Mutant Lung Cancer. Cancer Discov. 2022;12:2666–2683. doi: 10.1158/2159-8290.Cd-22-0111. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Matsuda T., Bebenek K., Masutani C., Hanaoka F., Kunkel T.A. Low fidelity DNA synthesis by human DNA polymerase-eta. Nature. 2000;404:1011–1013. doi: 10.1038/35010014. [DOI] [PubMed] [Google Scholar]
53.Agnihotri S., Burrell K., Buczkowicz P., Remke M., Golbourn B., Chornenkyy Y., Gajadhar A., Fernandez N.A., Clarke I.D., Barszczyk M.S., et al. ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents. Cancer Discov. 2014;4:1198–1213. doi: 10.1158/2159-8290.Cd-14-0157. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Xiao Q., McAtee C.K., Su X. Phase separation in immune signalling. Nat. Rev. Immunol. 2022;22:188–199. doi: 10.1038/s41577-021-00572-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Shi H., Wei J., He C. Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers. Mol. Cell. 2019;74:640–650. doi: 10.1016/j.molcel.2019.04.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Zhou C., Chen X., Planells-Cases R., Chu J., Wang L., Cao L., Li Z., López-Cayuqueo K.I., Xie Y., Ye S., et al. Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity. Immunity. 2020;52:767–781.e6. doi: 10.1016/j.immuni.2020.03.016. [DOI] [PubMed] [Google Scholar]
57.Wang L., Li S., Wang K., Wang N., Liu Q., Sun Z., Wang L., Wang L., Liu Q., Song C., Yang Q. Spermine enhances antiviral and anticancer responses by stabilizing DNA binding with the DNA sensor cGAS. Immunity. 2023;56:272–288.e7. doi: 10.1016/j.immuni.2023.01.001. [DOI] [PubMed] [Google Scholar]
58.Song C., Wang L., Wu X., Wang K., Xie D., Xiao Q., Li S., Jiang K., Liao L., Yates J.R., 3rd, et al. PML Recruits TET2 to Regulate DNA Modification and Cell Proliferation in Response to Chemotherapeutic Agent. Cancer Res. 2018;78:2475–2489. doi: 10.1158/0008-5472.CAN-17-3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Protter D.S.W., Parker R. Principles and Properties of Stress Granules. Trends Cell Biol. 2016;26:668–679. doi: 10.1016/j.tcb.2016.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S6 and Table S2

mmc1.pdf^{(2.1MB, pdf)}

Table S1. The candidate genes from meta analyses

mmc2.xlsx^{(30.1KB, xlsx)}

Document S2. Article plus supplemental information

mmc3.pdf^{(6.8MB, pdf)}

Data Availability Statement

[bib1] 1.Schlee M., Hartmann G. Discriminating self from non-self in nucleic acid sensing. Nat. Rev. Immunol. 2016;16:566–580. doi: 10.1038/nri.2016.78. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib2] 2.Bartok E., Hartmann G. Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids. Immunity. 2020;53:54–77. doi: 10.1016/j.immuni.2020.06.014. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Sun L., Wu J., Du F., Chen X., Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013;339:786–791. doi: 10.1126/science.1232458. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Wu J., Sun L., Chen X., Du F., Shi H., Chen C., Chen Z.J. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 2013;339:826–830. doi: 10.1126/science.1229963. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Li X., Shu C., Yi G., Chaton C.T., Shelton C.L., Diao J., Zuo X., Kao C.C., Herr A.B., Li P. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity. 2013;39:1019–1031. doi: 10.1016/j.immuni.2013.10.019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Andreeva L., Hiller B., Kostrewa D., Lässig C., de Oliveira Mann C.C., Jan Drexler D., Maiser A., Gaidt M., Leonhardt H., Hornung V., Hopfner K.P. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders. Nature. 2017;549:394–398. doi: 10.1038/nature23890. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Zhou W., Whiteley A.T., de Oliveira Mann C.C., Morehouse B.R., Nowak R.P., Fischer E.S., Gray N.S., Mekalanos J.J., Kranzusch P.J. Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance. Cell. 2018;174:300–311.e11. doi: 10.1016/j.cell.2018.06.026. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Du M., Chen Z.J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science. 2018;361:704–709. doi: 10.1126/science.aat1022. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9.Wion D., Casadesús J. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat. Rev. Microbiol. 2006;4:183–192. doi: 10.1038/nrmicro1350. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Schiffers S., Ebert C., Rahimoff R., Kosmatchev O., Steinbacher J., Bohne A.V., Spada F., Michalakis S., Nickelsen J., Müller M., Carell T. Quantitative LC-MS Provides No Evidence for m(6) dA or m(4) dC in the Genome of Mouse Embryonic Stem Cells and Tissues. Angew. Chem. Int. Ed. Engl. 2017;56:11268–11271. doi: 10.1002/anie.201700424. [DOI] [PubMed] [Google Scholar]

[bib11] 11.O'Brown Z.K., Boulias K., Wang J., Wang S.Y., O'Brown N.M., Hao Z., Shibuya H., Fady P.E., Shi Y., He C., et al. Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA. BMC Genom. 2019;20:445. doi: 10.1186/s12864-019-5754-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Fu Y., Luo G.Z., Chen K., Deng X., Yu M., Han D., Hao Z., Liu J., Lu X., Dore L.C., et al. N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell. 2015;161:879–892. doi: 10.1016/j.cell.2015.04.010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13.Greer E.L., Blanco M.A., Gu L., Sendinc E., Liu J., Aristizábal-Corrales D., Hsu C.H., Aravind L., He C., Shi Y. DNA Methylation on N6-Adenine in C. elegans. Cell. 2015;161:868–878. doi: 10.1016/j.cell.2015.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Zhang G., Huang H., Liu D., Cheng Y., Liu X., Zhang W., Yin R., Zhang D., Zhang P., Liu J., et al. N6-methyladenine DNA modification in Drosophila. Cell. 2015;161:893–906. doi: 10.1016/j.cell.2015.04.018. [DOI] [PubMed] [Google Scholar]

[bib15] 15.Krüger D.H., Bickle T.A. Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts. Microbiol. Rev. 1983;47:345–360. doi: 10.1128/mr.47.3.345-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16.Dominissini D., Moshitch-Moshkovitz S., Schwartz S., Salmon-Divon M., Ungar L., Osenberg S., Cesarkas K., Jacob-Hirsch J., Amariglio N., Kupiec M., et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485:201–206. doi: 10.1038/nature11112. [DOI] [PubMed] [Google Scholar]

[bib17] 17.Gao Y., Pei G., Li D., Li R., Shao Y., Zhang Q.C., Li P. Multivalent m(6)A motifs promote phase separation of YTHDF proteins. Cell Res. 2019;29:767–769. doi: 10.1038/s41422-019-0210-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Ries R.J., Zaccara S., Klein P., Olarerin-George A., Namkoong S., Pickering B.F., Patil D.P., Kwak H., Lee J.H., Jaffrey S.R. m(6)A enhances the phase separation potential of mRNA. Nature. 2019;571:424–428. doi: 10.1038/s41586-019-1374-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19.Cheng Y., Xie W., Pickering B.F., Chu K.L., Savino A.M., Yang X., Luo H., Nguyen D.T., Mo S., Barin E., et al. N(6)-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation. Cancer Cell. 2021;39:958–972.e8. doi: 10.1016/j.ccell.2021.04.017. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20.Lee J.H., Wang R., Xiong F., Krakowiak J., Liao Z., Nguyen P.T., Moroz-Omori E.V., Shao J., Zhu X., Bolt M.J., et al. Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation. Mol. Cell. 2021;81:3368–3385.e9. doi: 10.1016/j.molcel.2021.07.024. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Hu S., Sun H., Yin L., Li J., Mei S., Xu F., Wu C., Liu X., Zhao F., Zhang D., et al. PKR-dependent cytosolic cGAS foci are necessary for intracellular DNA sensing. Sci. Signal. 2019;12 doi: 10.1126/scisignal.aav7934. [DOI] [PubMed] [Google Scholar]

[bib22] 22.Liu Z.S., Cai H., Xue W., Wang M., Xia T., Li W.J., Xing J.Q., Zhao M., Huang Y.J., Chen S., et al. G3BP1 promotes DNA binding and activation of cGAS. Nat. Immunol. 2019;20:18–28. doi: 10.1038/s41590-018-0262-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Su R., Dong L., Li Y., Gao M., Han L., Wunderlich M., Deng X., Li H., Huang Y., Gao L., et al. Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion. Cancer Cell. 2020;38:79–96.e11. doi: 10.1016/j.ccell.2020.04.017. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 24.Abakir A., Giles T.C., Cristini A., Foster J.M., Dai N., Starczak M., Rubio-Roldan A., Li M., Eleftheriou M., Crutchley J., et al. N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat. Genet. 2020;52:48–55. doi: 10.1038/s41588-019-0549-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 25.Zhang C., Chen L., Peng D., Jiang A., He Y., Zeng Y., Xie C., Zhou H., Luo X., Liu H., et al. METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation. Mol. Cell. 2020;79:425–442. doi: 10.1016/j.molcel.2020.06.017. [DOI] [PubMed] [Google Scholar]

[bib26] 26.Lauring A.S., Frydman J., Andino R. The role of mutational robustness in RNA virus evolution. Nat. Rev. Microbiol. 2013;11:327–336. doi: 10.1038/nrmicro3003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib27] 27.Warren T.K., Jordan R., Lo M.K., Ray A.S., Mackman R.L., Soloveva V., Siegel D., Perron M., Bannister R., Hui H.C., et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016;531:381–385. doi: 10.1038/nature17180. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib28] 28.Wang L., Song C., Wang N., Li S., Liu Q., Sun Z., Wang K., Yu S.C., Yang Q. NADP modulates RNA m(6)A methylation and adipogenesis via enhancing FTO activity. Nat. Chem. Biol. 2020;16:1394–1402. doi: 10.1038/s41589-020-0601-2. [DOI] [PubMed] [Google Scholar]

[bib29] 29.Jia G., Fu Y., Zhao X., Dai Q., Zheng G., Yang Y., Yi C., Lindahl T., Pan T., Yang Y.G., He C. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 2011;7:885–887. doi: 10.1038/nchembio.687. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 30.Yankova E., Blackaby W., Albertella M., Rak J., De Braekeleer E., Tsagkogeorga G., Pilka E.S., Aspris D., Leggate D., Hendrick A.G., et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021;593:597–601. doi: 10.1038/s41586-021-03536-w. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Li X.D., Wu J., Gao D., Wang H., Sun L., Chen Z.J. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science. 2013;341:1390–1394. doi: 10.1126/science.1244040. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32.Vincent J., Adura C., Gao P., Luz A., Lama L., Asano Y., Okamoto R., Imaeda T., Aida J., Rothamel K., et al. Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice. Nat. Commun. 2017;8:750. doi: 10.1038/s41467-017-00833-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib59] 33.Youn J.Y., Dyakov B.J.A., Zhang J., Knight J.D.R., Vernon R.M., Forman-Kay J.D., Gingras A.C. Properties of Stress Granule and P-Body Proteomes. Mol. Cell. 2019;76:286–294. doi: 10.1016/j.molcel.2019.09.014. [DOI] [PubMed] [Google Scholar]

[bib33] 34.Gilbert W.V., Bell T.A., Schaening C. Messenger RNA modifications: Form, distribution, and function. Science. 2016;352:1408–1412. doi: 10.1126/science.aad8711. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 35.Luecke S., Holleufer A., Christensen M.H., Jønsson K.L., Boni G.A., Sørensen L.K., Johannsen M., Jakobsen M.R., Hartmann R., Paludan S.R. cGAS is activated by DNA in a length-dependent manner. EMBO Rep. 2017;18:1707–1715. doi: 10.15252/embr.201744017. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 36.Li D., Delaney J.C., Page C.M., Yang X., Chen A.S., Wong C., Drennan C.L., Essigmann J.M. Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB. J. Am. Chem. Soc. 2012;134:8896–8901. doi: 10.1021/ja3010094. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 37.Aughey G.N., Cheetham S.W., Southall T.D. DamID as a versatile tool for understanding gene regulation. Development. 2019;146 doi: 10.1242/dev.173666. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 38.Zhang Y., Nelson M., Nietfeldt J., Xia Y., Burbank D., Ropp S., Van Etten J.L. Chlorella virus NY-2A encodes at least 12 DNA endonuclease/methyltransferase genes. Virology. 1998;240:366–375. doi: 10.1006/viro.1997.8936. [DOI] [PubMed] [Google Scholar]

[bib38] 39.Mehellou Y., Rattan H.S., Balzarini J. The ProTide Prodrug Technology: From the Concept to the Clinic. J. Med. Chem. 2018;61:2211–2226. doi: 10.1021/acs.jmedchem.7b00734. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] 40.Frye M., Harada B.T., Behm M., He C. RNA modifications modulate gene expression during development. Science. 2018;361:1346–1349. doi: 10.1126/science.aau1646. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib40] 41.Blow M.J., Clark T.A., Daum C.G., Deutschbauer A.M., Fomenkov A., Fries R., Froula J., Kang D.D., Malmstrom R.R., Morgan R.D., et al. The Epigenomic Landscape of Prokaryotes. Plos Genet. 2016;12 doi: 10.1371/journal.pgen.1005854. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib41] 42.Wang L., Wen M., Cao X. Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science. 2019;365 doi: 10.1126/science.aav0758. [DOI] [PubMed] [Google Scholar]

[bib42] 43.Alarcón C.R., Goodarzi H., Lee H., Liu X., Tavazoie S., Tavazoie S.F. HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell. 2015;162:1299–1308. doi: 10.1016/j.cell.2015.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 44.Arguello A.E., DeLiberto A.N., Kleiner R.E. RNA Chemical Proteomics Reveals the N(6)-Methyladenosine (m(6)A)-Regulated Protein-RNA Interactome. J. Am. Chem. Soc. 2017;139:17249–17252. doi: 10.1021/jacs.7b09213. [DOI] [PubMed] [Google Scholar]

[bib44] 45.Ablasser A., Chen Z.J. cGAS in action: Expanding roles in immunity and inflammation. Science. 2019;363 doi: 10.1126/science.aat8657. [DOI] [PubMed] [Google Scholar]

[bib45] 46.Roers A., Hiller B., Hornung V. Recognition of Endogenous Nucleic Acids by the Innate Immune System. Immunity. 2016;44:739–754. doi: 10.1016/j.immuni.2016.04.002. [DOI] [PubMed] [Google Scholar]

[bib46] 47.Petroni G., Buqué A., Zitvogel L., Kroemer G., Galluzzi L. Immunomodulation by targeted anticancer agents. Cancer Cell. 2021;39:310–345. doi: 10.1016/j.ccell.2020.11.009. [DOI] [PubMed] [Google Scholar]

[bib47] 48.Kwon J., Bakhoum S.F. The Cytosolic DNA-Sensing cGAS-STING Pathway in Cancer. Cancer Discov. 2020;10:26–39. doi: 10.1158/2159-8290.CD-19-0761. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib48] 49.De Clercq E. Strategies in the design of antiviral drugs. Nat. Rev. Drug Discov. 2002;1:13–25. doi: 10.1038/nrd703. [DOI] [PubMed] [Google Scholar]

[bib49] 50.Bekerman E., Einav S. Infectious disease. Combating emerging viral threats. Science. 2015;348:282–283. doi: 10.1126/science.aaa3778. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib50] 51.Noronha A., Belugali Nataraj N., Lee J.S., Zhitomirsky B., Oren Y., Oster S., Lindzen M., Mukherjee S., Will R., Ghosh S., et al. AXL and Error-Prone DNA Replication Confer Drug Resistance and Offer Strategies to Treat EGFR-Mutant Lung Cancer. Cancer Discov. 2022;12:2666–2683. doi: 10.1158/2159-8290.Cd-22-0111. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] 52.Matsuda T., Bebenek K., Masutani C., Hanaoka F., Kunkel T.A. Low fidelity DNA synthesis by human DNA polymerase-eta. Nature. 2000;404:1011–1013. doi: 10.1038/35010014. [DOI] [PubMed] [Google Scholar]

[bib52] 53.Agnihotri S., Burrell K., Buczkowicz P., Remke M., Golbourn B., Chornenkyy Y., Gajadhar A., Fernandez N.A., Clarke I.D., Barszczyk M.S., et al. ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents. Cancer Discov. 2014;4:1198–1213. doi: 10.1158/2159-8290.Cd-14-0157. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib53] 54.Xiao Q., McAtee C.K., Su X. Phase separation in immune signalling. Nat. Rev. Immunol. 2022;22:188–199. doi: 10.1038/s41577-021-00572-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib54] 55.Shi H., Wei J., He C. Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers. Mol. Cell. 2019;74:640–650. doi: 10.1016/j.molcel.2019.04.025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib55] 56.Zhou C., Chen X., Planells-Cases R., Chu J., Wang L., Cao L., Li Z., López-Cayuqueo K.I., Xie Y., Ye S., et al. Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity. Immunity. 2020;52:767–781.e6. doi: 10.1016/j.immuni.2020.03.016. [DOI] [PubMed] [Google Scholar]

[bib56] 57.Wang L., Li S., Wang K., Wang N., Liu Q., Sun Z., Wang L., Wang L., Liu Q., Song C., Yang Q. Spermine enhances antiviral and anticancer responses by stabilizing DNA binding with the DNA sensor cGAS. Immunity. 2023;56:272–288.e7. doi: 10.1016/j.immuni.2023.01.001. [DOI] [PubMed] [Google Scholar]

[bib57] 58.Song C., Wang L., Wu X., Wang K., Xie D., Xiao Q., Li S., Jiang K., Liao L., Yates J.R., 3rd, et al. PML Recruits TET2 to Regulate DNA Modification and Cell Proliferation in Response to Chemotherapeutic Agent. Cancer Res. 2018;78:2475–2489. doi: 10.1158/0008-5472.CAN-17-3091. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib58] 59.Protter D.S.W., Parker R. Principles and Properties of Stress Granules. Trends Cell Biol. 2016;26:668–679. doi: 10.1016/j.tcb.2016.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Host RNA N6-methyladenosine and incoming DNA N6-methyldeoxyadenosine modifications cooperatively elevate the condensation potential of DNA to activate immune surveillance

Na Wang

Qiaoling Liu

Bo Wang

Zhuo Yang

Siru Li

Ran Li

Xinyuan Liang

Jiayu Fan

Hui Wang

Zhen Sun

Ling Dong

Yueru Hou

Shengnan Wang

Chengli Song

Yang Wang

Chunshan Quan

Qingkai Yang

Lina Wang