Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

GDF15 Protects Insulin-Producing Beta Cells against Pro-Inflammatory Cytokines and Metabolic Stress via Increased Deamination of Intracellular Adenosine.

It has been proposed that antidiabetic drugs, such as metformin and imatinib, at least in part, promote improved glucose tolerance in type 2 diabetic patients via increased production of the inflammatory cytokine GDF15. This is supported by studies, performed in rodent cell lines and mouse models, in which the addition or production of GDF15 improved beta-cell function and survival. The aim of the present study was to determine whether human beta cells produce GDF15 in response to antidiabetic drugs and, if so, to further elucidate the mechanisms by which GDF15 modulates the function and survival of such cells. The effects and expression of GDF15 were analyzed in human insulin-producing EndoC-betaH1 cells and human islets. We observed that alpha and beta cells exhibit considerable heterogeneity in GDF15 immuno-positivity. The predominant form of GDF15 present in islet and EndoC-betaH1 cells was pro-GDF15. Imatinib, but not metformin, increased pro-GDF15 levels in EndoC-betaH1 cells. Under basal conditions, exogenous GDF15 increased human islet oxygen consumption rates. In EndoC-betaH1 cells and human islets, exogenous GDF15 partially ameliorated cytokine- or palmitate + high-glucose-induced loss of function and viability. GDF15-induced cell survival was paralleled by increased inosine levels, suggesting a more efficient disposal of intracellular adenosine. Knockdown of adenosine deaminase, the enzyme that converts adenosine to inosine, resulted in lowered inosine levels and loss of protection against cytokine- or palmitate + high-glucose-induced cell death. It is concluded that imatinib-induced GDF15 production may protect human beta cells partially against inflammatory and metabolic stress. Furthermore, it is possible that the GDF15-mediated activation of adenosine deaminase and the increased disposal of intracellular adenosine participate in protection against beta-cell death.

High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma.

Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5' side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genes RPL13A and RPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription.

Analysis of cytosine deamination events in excision repair sequencing reads reveals mechanisms of incision site selection in NER.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions and is therefore critical for genome stability. During NER, DNA is unwound on either side of the lesion and excised, but the rules governing incision site selection, particularly in eukaryotic cells, are unclear. Excision repair-sequencing (XR-seq) sequences excised NER fragments, but analysis has been limited because the lesion location is unknown. Here, we exploit accelerated cytosine deamination rates in UV-induced CPD (cyclobutane pyrimidine dimer) lesions to precisely map their locations at C to T mismatches in XR-seq reads, revealing general and species-specific patterns of incision site selection during NER. Our data indicate that the 5' incision site occurs preferentially in HYV (i.e. not G; C/T; not T) sequence motifs, a pattern that can be explained by sequence preferences of the XPF-ERCC1 endonuclease. In contrast, the 3' incision site does not show strong sequence preferences, once truncated reads arising from mispriming events are excluded. Instead, the 3' incision is partially determined by the 5' incision site distance, indicating that the two incision events are coupled. Finally, our data reveal unique and coupled NER incision patterns at nucleosome boundaries. These findings reveal key principles governing NER incision site selection in eukaryotic cells.

Deamination-triggered exponential signal amplification for chemiluminescent detection of cytosine deaminase at the single-cell level.

We construct a sensitive chemiluminescent biosensor for sensitive detection of cytosine deaminase APOBEC3A based on deamination-triggered exponential signal amplification. This biosensor displays good specificity and high sensitivity, and it can screen APOBEC3A inhibitors and measure endogenous APOBEC3A at the single-cell level, with prospective applications in disease diagnostics and therapy.

Single-Base Resolution Detection of N6-Methyladenosine in RNA by Adenosine Deamination Sequencing.

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.

Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA.

The interbacterial deaminase toxin DddA catalyzes cytosine-to-uracil conversion in double-stranded (ds) DNA and enables CRISPR-free mitochondrial base editing, but the molecular mechanisms underlying its unique substrate selectivity have remained elusive. Here, we report crystal structures of DddA bound to a dsDNA substrate containing the 5'-TC target motif. These structures show that DddA binds to the minor groove of a sharply bent dsDNA and engages the target cytosine extruded from the double helix. DddA Phe1375 intercalates in dsDNA and displaces the 5' (-1) thymine, which in turn replaces the target (0) cytosine and forms a noncanonical T-G base pair with the juxtaposed guanine. This tandem displacement mechanism allows DddA to locate a target cytosine without flipping it into the active site. Biochemical experiments demonstrate that DNA base mismatches enhance the DddA deaminase activity and relax its sequence selectivity. On the basis of the structural information, we further identified DddA mutants that exhibit attenuated activity or altered substrate preference. Our studies may help design new tools useful in genome editing or other applications.

Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA.

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site 'HxE-PCxxC' and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

DNA Deamination Is Required for Human APOBEC3A-Driven Hepatocellular Carcinoma In Vivo.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases is well-known for its antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3's signature single-base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as utilized in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA-editing activity is defective in promoting tumor formation. Collectively, these results demonstrate that APOBEC3A is a "master driver" that fuels tumor formation through a DNA deamination-dependent mechanism.

Apobec3A Deamination Functions Are Involved in Antagonizing Efficient Human Adenovirus Replication and Gene Expression.

Apobec3A is involved in the antiviral host defense, targeting nuclear DNA, introducing point mutations, and thereby activating DNA damage response (DDR). Here, we found a significant upregulation of Apobec3A during HAdV infection, including Apobec3A protein stabilization mediated by the viral proteins E1B-55K and E4orf6, which subsequently limited HAdV replication and most likely involved a deaminase-dependent mechanism. The transient silencing of Apobec3A enhanced adenoviral replication. HAdV triggered Apobec3A dimer formation and enhanced activity to repress the virus. Apobec3A decreased E2A SUMOylation and interfered with viral replication centers. A comparative sequence analysis revealed that HAdV types A, C, and F may have evolved a strategy to escape Apobec3A-mediated deamination via reduced frequencies of TC dinucleotides within the viral genome. Although viral components induce major changes within infected cells to support lytic life cycles, our findings demonstrate that host Apobec3A-mediated restriction limits virus replication, albeit that HAdV may have evolved to escape this restriction. This allows for novel insights into the HAdV/host-cell interplay, which broaden the current view of how a host cell can limit HAdV infection. IMPORTANCE Our data provide a novel conceptual insight into the virus/host-cell interplay, changing the current view of how a host-cell can defeat a virus infection. Thus, our study reveals a novel and general impact of cellular Apobec3A on the intervention of human adenovirus (HAdV) gene expression and replication by improving the host antiviral defense mechanisms, thereby providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways that are modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as COVID vaccine vectors and also frequently serve as tools in human gene therapy and oncolytic treatment options. HAdV constitute an ideal model system by which to analyze the transforming capabilities of DNA tumor viruses as well as the underlying molecular principles of virus-induced and cellular tumorigenesis.

Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast.

Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.

Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination.

N6-methyladenosine (m6A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m6A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m6A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m6A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m6A detection from limited input samples. In addition to transcriptome-wide m6A profiling, we demonstrate site-specific, deep-sequencing-free m6A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m6A landscape at unprecedented resolution, but also detect m6A at user-specified loci with a simple workflow.

C-to-U RNA deamination is the driving force accelerating SARS-CoV-2 evolution.

Understanding the molecular mechanism underlying the rampant mutation of SARS-CoV-2 would help us control the COVID-19 pandemic. The APOBEC-mediated C-to-U deamination is a major mutation type in the SARS-CoV-2 genome. However, it is unclear whether the novel mutation rate u is higher for C-to-U than for other mutation types, and what the detailed driving force is. By analyzing the time course SARS-CoV-2 global population data, we found that C-to-U has the highest novel mutation rate u among all mutation types and that this u is still increasing with time (du/dt > 0). Novel C-to-U events, rather than other mutation types, have a preference over particular genomic regions. A less local RNA structure is correlated with a high novel C-to-U mutation rate. A cascade model nicely explains the du/dt > 0 for C-to-U deamination. In SARS-CoV-2, the RNA structure serves as the molecular basis of the extremely high and continuously accelerating C-to-U deamination rate. This mechanism is the driving force of the mutation, adaptation, and evolution of SARS-CoV-2. Our findings help us understand the dynamic evolution of the virus mutation rate.

Deamination of 1-Aminoalkylphosphonic Acids: Reaction Intermediates and Selectivity.

Deamination of 1-aminoalkylphosphonic acids in the reaction with HNO2 (generated "in situ" from NaNO2) yields a mixture of substitution products (1-hydroxyalkylphosphonic acids), elimination products (vinylphosphonic acid derivatives), rearrangement and substitution products (2-hydroxylkylphosphonic acids) as well as H3PO4. The variety of formed reaction products suggests that 1-phosphonoalkylium ions may be intermediates in such deamination reactions.

Serine Deamination Is a New Acid Tolerance Mechanism Observed in Uropathogenic Escherichia coli.

Escherichia coli associates with humans early in life and can occupy several body niches either as a commensal in the gut and vagina, or as a pathogen in the urinary tract. As such, E. coli has an arsenal of acid response mechanisms that allow it to withstand the different levels of acid stress encountered within and outside the host. Here, we report the discovery of an additional acid response mechanism that involves the deamination of l-serine to pyruvate by the conserved l-serine deaminases SdaA and SdaB. l-serine is the first amino acid to be imported in E. coli during growth in laboratory media. However, there remains a lack in knowledge as to how l-serine is utilized. Using a uropathogenic strain of E. coli, UTI89, we show that in acidified media, l-serine is brought into the cell via the SdaC transporter. We further demonstrate that deletion of the l-serine deaminases SdaA and SdaB renders E. coli susceptible to acid stress, similar to other acid stress deletion mutants. The pyruvate produced by l-serine deamination activates the pyruvate sensor BtsS, which in concert with the noncognate response regulator YpdB upregulates the putative transporter YhjX. Based on these observations, we propose that l-serine deamination constitutes another acid response mechanism in E. coli. IMPORTANCE The observation that l-serine uptake occurs as E. coli cultures grow is well established, yet the benefit E. coli garners from this uptake remains unclear. Here, we report a novel acid tolerance mechanism where l-serine is deaminated to pyruvate and ammonia, promoting survival of E. coli under acidic conditions. This study is important as it provides evidence of the use of l-serine as an acid response strategy, not previously reported for E. coli.

Hanseniaspora uvarum FS35 degrade putrescine in wine through the direct oxidative deamination pathway of copper amine oxidase 1.

Putrescine is abundant in wine and have toxicological risks for the health of consumers. Certain microbes with oxidative deamination activity are considered to be one of the most effective ways to degrade putrescine. The characterization and possible mechanism of putrescine degradation by Hanseniaspora uvarum FS35 were studied in this work. Hanseniaspora uvarum FS35 was selected from 111 yeast strains by UPLC analysis and exhibited the ability to eliminate > 44.5 mg/L of putrescine after 12 h of culture. Transcriptome analysis showed that by adding putrescine as a nitrogen source, the gene expression level of copper amine oxidase 1 (CuAO1) increased, leading to a coordinated response in the oxidative deamination of putrescine to 4-amino-butanal and subsequent dehydrogenation to 4-amino-butanoate. The purified recombinant protein CuAO1 could degrade 25.8 and 21.8 mg/L of putrescine in Marselan and Cabernet Sauvignon wines, respectively. H. uvarum FS35 was then inoculated sequentially with Saccharomyces cerevisiae into Cabernet Sauvignon grape juice, and the physiochemical indexes and aroma compounds were detected by HPLC and HS-SPME/GC-MS, respectively. wines produced from sequential inoculations showed significantly lower level of putrescine and higher amounts of glycerol, lactic acid, acetic acid, phenylethyl alcohol, ethyl acetate and ß-phenylethyl acetate compared with the control fermentation of commercial S. cerevisiae, which proved the potential of H. uvarum FS35 as a promising strategy to reduce biogenic amines in wines.

Competition for DNA binding between the genome protector replication protein A and the genome modifying APOBEC3 single-stranded DNA deaminases.

The human APOBEC family of eleven cytosine deaminases use RNA and single-stranded DNA (ssDNA) as substrates to deaminate cytosine to uracil. This deamination event has roles in lipid metabolism by altering mRNA coding, adaptive immunity by causing evolution of antibody genes, and innate immunity through inactivation of viral genomes. These benefits come at a cost where some family members, primarily from the APOBEC3 subfamily (APOBEC3A-H, excluding E), can cause off-target deaminations of cytosine to form uracil on transiently single-stranded genomic DNA, which induces mutations that are associated with cancer evolution. Since uracil is only promutagenic, the mutations observed in cancer genomes originate only when uracil is not removed by uracil DNA glycosylase (UNG) or when the UNG-induced abasic site is erroneously repaired. However, when ssDNA is present, replication protein A (RPA) binds and protects the DNA from nucleases or recruits DNA repair proteins, such as UNG. Thus, APOBEC enzymes must compete with RPA to access their substrate. Certain APOBEC enzymes can displace RPA, bind and scan ssDNA efficiently to search for cytosines, and can become highly overexpressed in tumor cells. Depending on the DNA replication conditions and DNA structure, RPA can either be in excess or deficient. Here we discuss the interplay between these factors and how despite RPA, multiple cancer genomes have a mutation bias at cytosines indicative of APOBEC activity.

Tandem Reduction, Ammonolysis, Condensation, and Deamination Reaction for Synthesis of Benzothiadiazines and 1-(Phenylsulfonyl)-1H-benzimidazoles.

A novel route for a SnCl2-promoted tandem reduction, ammonolysis, condensation, and deamination reaction which uses nitrile and 2-nitro-N-phenylbenzenesulfonamide/N-(2-nitrophenyl)benzenesulfonamide to synthesize derivatives of benzothiadiazine/1-(phenylsulfonyl)-1H-benzimidazole has been developed. The method features convenient operation and good functional group tolerance. In addition, it employs unsensitive and inexpensive SnCl2/i-PrOH as the reaction reagent and provides a direct approach for the synthesis of pharmaceutically important targets.

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing.

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor ß (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA.

Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.