A hybrid residue based sequential encoding mechanism with XGBoost improved ensemble model for identifying 5-hydroxymethylcytosine modifications.

RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA's operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis.

A disposable electrochemical magnetic immunosensor for the rapid and sensitive detection of 5-formylcytosine and 5-carboxylcytosine in DNA.

5-formylcytosine (5 fC) and 5-carboxylcytosine (5caC) serve as key intermediates in DNA demethylation process with significant implications for gene regulation and disease progression. In this study, we introduce a novel electrochemical sensing platform specifically designed for the sensitive and selective detection of 5 fC and 5caC in DNA. Protein A-modified magnetic beads (ProtA-MBs) coupled with specific antibodies facilitate the immunorecognition and enrichment of these modified bases. Signal amplification is achieved through several chemical reactions involving the interaction between N3-kethonaxl and guanine, copper-free click chemistry for the attachment of dibenzocyclooctyne (DBCO)-Biotin, and the subsequent recognition by streptavidin-conjugated horseradish peroxidase (SA-HRP). The assay's readout is performed on a disposable laser-induced graphene (LIG) electrode, modified with the bead-antibody-DNA complex in a magnetic field, and analyzed using differential pulse voltammetry in a system employing hydroquinone (HQ) as the redox mediator and H2O2 as the substrate. This immunosensor displayed excellent sensitivity, with detection limits of 14.8 fM for 5 fC across a 0.1-1000 pM linear range and 87.4 fM for 5caC across a 0.5-5000 pM linear range, and maintained high selectivity even in the presence of interferences from other DNA modifications. Successful application in quantifying 5 fC and 5caC in genomic DNA from cell extracts, with recovery rates between 97.7% to 102.9%, underscores its potential for clinical diagnostics. N3-kethoxal was used for the first time in an electrochemical sensor. This work not only broadens the toolkit for detecting DNA modifications but also provides a fresh impetus for the development of point-of-care testing (POCT) technologies.

Methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Cytosine analogues as DNA methyltransferase substrates.

DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML) and possibly ß-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC) and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases.

Brincidofovir inhibits polyomavirus infection in vivo.

Polyomaviruses are species-specific DNA viruses that can cause disease in immunocompromised individuals. Despite their role as the causative agents for several diseases, there are no currently approved antivirals for treating polyomavirus infection. Brincidofovir (BCV) is an antiviral approved for the treatment of poxvirus infections and has shown activity against other double-stranded DNA viruses. In this study, we tested the efficacy of BCV against polyomavirus infection in vitro and in vivo using mouse polyomavirus (MuPyV). BCV inhibited virus production in primary mouse kidney cells and brain cortical cells. BCV treatment of cells transfected with MuPyV genomic DNA resulted in a reduction in virus levels, indicating that viral inhibition occurs post-entry. Although in vitro BCV treatment had a limited effect on viral DNA and RNA levels, drug treatment was associated with a reduction in viral protein, raising the possibility that BCV acts post-transcriptionally to inhibit MuPyV infection. In mice, BCV treatment was well tolerated, and prophylactic treatment resulted in a reduction in viral DNA levels and a potent suppression of infectious virus production in the kidney and brain. In mice with chronic polyomavirus infection, therapeutic administration of BCV decreased viremia and reduced infection in the kidney. These data demonstrate that BCV exerts antiviral activity against polyomavirus infection in vivo, supporting further investigation into the use of BCV to treat clinical polyomavirus infections. IMPORTANCE: Widespread in the human population and able to persist asymptomatically for the life of an individual, polyomavirus infections cause a significant disease burden in the immunocompromised. Individuals undergoing immune suppression, such as kidney transplant patients or those treated for autoimmune diseases, are particularly at high risk for polyomavirus-associated diseases. Because no antiviral agent exists for treating polyomavirus infections, management of polyomavirus-associated diseases typically involves reducing or discontinuing immunomodulatory therapy. This can be perilous due to the risk of transplant rejection and the potential development of adverse immune reactions. Thus, there is a pressing need for the development of antivirals targeting polyomaviruses. Here, we investigate the effects of brincidofovir, an FDA-approved antiviral, on polyomavirus infection in vivo using mouse polyomavirus. We show that the drug is well-tolerated in mice, reduces infectious viral titers, and limits viral pathology, indicating the potential of brincidofovir as an anti-polyomavirus therapeutic.

Bioinspired Heterocoordination in Adaptable Cobalt Metal-Organic Framework for DNA Epigenetic Modification Detection.

Metal-organic frameworks (MOFs) show unique advantages in simulating the dynamics and fidelity of natural coordination. Inspired by zinc finger protein, a second linker was introduced to affect the homogeneous MOF system and thus facilitate the emergence of diverse functionalities. Under the systematic identification of 12 MOF species (i.e., metal ions, linkers) and 6 second linkers (trigger), a dissipative system consisting of Co-BDC-NO2 and o-phenylenediamine (oPD) was screened out, which can rapidly and in situ generate a high photothermal complex (η = 36.9%). Meanwhile, both the carboxylation of epigenetic modifications and metal ion (Fe3+, Ni2+, Cu2+, Zn2+, Co2+ and Mn2+) screening were utilized to improve the local coordination environment so that the adaptable Co-MOF growth on the DNA strand was realized. Thus, epigenetic modification information on DNA was converted to an amplified metal ion signal, and then oPD was further introduced to generate bimodal dissipative signals by which a simple, high-sensitivity detection strategy of 5-hydroxymethylcytosine (LOD = 0.02%) and 5-formylcytosine (LOD = 0.025‰) was developed. The strategy provides one low-cost method (< 0.01 $/sample) for quantifying global epigenetic modifications, which greatly promotes epigenetic modification-based early disease diagnosis. This work also proposes a general heterocoordination design concept for molecular recognition and signal transduction, opening a new MOF-based sensing paradigm.

Theoretical Insights into N-Glycoside Bond Cleavage of 5-Carboxycytosine by Thymine DNA Glycosylase: A QM/MM Study.

Thymine DNA glycosylase (TDG)-mediated excision of 5-formylcytosine and 5-carboxylcytosine (5-caC) is a critical step in active DNA demethylation. Herein, we employed a combined quantum mechanics/molecular mechanics approach to investigate the reaction mechanism of TDG-catalyzed N-glycosidic bond cleavage of 5-caC. The calculated results show that TDG-catalyzed 5-caC excision follows a concerted (SN2) mechanism in which glycosidic bond dissociation is coupled with nucleophile attack. Protonation of the 5-caC anion contributes to the cleavage of the N-glycoside bond, in which the N3-protonated zwitterion and imino tautomers are more favorable than carboxyl-protonated amino tautomers. This is consistent with the experimental data. Furthermore, our results reveal that the configuration rearrangement process of the protonated 5-caC would lower the stability of the N-glycoside bond and substantially reduce the barrier height for the subsequent C1'-N1 bond cleavage. This should be attributed to the smaller electrostatic repulsion between the leaving base and the negative phosphate group as a result of the structural rearrangement.

NPP-669, a prodrug of cidofovir, is highly efficacious against human adenovirus infection in the permissive Syrian hamster model.

Human adenoviruses can cause serious, disseminated infections in immunocompromised patients. For pediatric allogeneic stem cell transplant patients, the case fatality rate can reach 80%. Still, there is no available antiviral drug that is specifically approved by the Food and Drug Administration for the treatment of adenovirus infections. To fill this pressing medical need, we have developed NPP-669, a prodrug of cidofovir with broad activity against double-stranded DNA viruses, including adenoviruses. Here, we report on the in vivo anti-adenoviral efficacy of NPP-669. Using the immunosuppressed Syrian hamster as the model, we show that NPP-669 is highly efficacious when dosed orally at 1 mg/kg and 3 mg/kg. In a delayed administration experiment, NPP-669 was more effective than brincidofovir, a similar compound that reached Phase III clinical trials. Furthermore, parenteral administration of NPP-669 increased its efficacy approximately 10-fold compared to oral dosing without apparent toxicity, suggesting that this route may be preferable in a hospital setting. Based on these findings, we believe that NPP-669 is a promising new compound that needs to be further investigated.

Computational analysis of antiviral drugs using topological descriptors.

Many health challenges are attributed to viral infections, which represent significant concerns in public health. Among these infections, diseases such as herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella-zoster virus (VZV) infections have garnered attention due to their prevalence and impact on human health. There are specific antiviral medications available for the treatment of these viral infections. Drugs like Cidofovir, Valacyclovir, and Acyclovir are commonly prescribed. These antiviral drugs are known for their efficacy against herpesviruses and related viral infections, leveraging their ability to inhibit viral DNA polymerase. A molecular descriptor is a numerical value that correlates with specific physicochemical properties of a molecular graph. This article explores the calculation of distance-based topological descriptors, including the Trinajstic, Mostar, Szeged, and PI descriptors for the aforementioned antiviral drugs. These descriptors provide insights into these drugs' structural and physicochemical characteristics, aiding in understanding their mechanism of action and the development of new therapeutic agents.

Treatment and Vaccination for Smallpox and Monkeypox.

The smallpox infection with the variola virus was one of the most fatal disorders until a global eradication was initiated in the twentieth century. The last cases were reported in Somalia 1977 and as a laboratory infection in the UK 1978; in 1980, the World Health Organization (WHO) declared smallpox for extinct. The smallpox virus with its very high transmissibility and mortality is still a major biothreat, because the vaccination against smallpox was stopped globally in the 1980s. For this reason, new antivirals (cidofovir, brincidofovir, and tecovirimat) and new vaccines (ACAM2000, LC16m8 and Modified Vaccine Ankara MVA) were developed. For passive immunization, vaccinia immune globulin intravenous (VIGIV) is available. Due to the relationships between orthopox viruses such as vaccinia, variola, mpox (monkeypox), cowpox, and horsepox, the vaccines (LC16m8 and MVA) and antivirals (brincidofovir and tecovirimat) could also be used in the mpox outbreak with positive preliminary data. As mutations can result in drug resistance against cidofovir or tecovirimat, there is need for further research. Further antivirals (NIOCH-14 and ST-357) and vaccines (VACΔ6 and TNX-801) are being developed in Russia and the USA. In conclusion, further research for treatment and prevention of orthopox infections is needed and is already in progress. After a brief introduction, this chapter presents the smallpox and mpox disease and thereafter full overviews on antiviral treatment and vaccination including the passive immunization with vaccinia immunoglobulins.

Structural basis of human mpox viral DNA replication inhibition by brincidofovir and cidofovir.

Mpox virus has wildly spread over 108 non-endemic regions in the world since May 2022. DNA replication of mpox is performed by DNA polymerase machinery F8-A22-E4, which is known as a great drug target. Brincidofovir and cidofovir are reported to have broad-spectrum antiviral activity against poxviruses, including mpox virus in animal models. However, the molecular mechanism is not understood. Here we report cryogenic electron microscopy structures of mpox viral F8-A22-E4 in complex with a DNA duplex, or dCTP and the DNA duplex, or cidofovir diphosphate and the DNA duplex at resolution of 3.22, 2.98 and 2.79 Å, respectively. Our structural work and DNA replication inhibition assays reveal that cidofovir diphosphate is located at the dCTP binding position with a different conformation to compete with dCTP to incorporate into the DNA and inhibit DNA synthesis. Conformation of both F8-A22-E4 and DNA is changed from the pre-dNTP binding state to DNA synthesizing state after dCTP or cidofovir diphosphate is bound, suggesting a coupling mechanism. This work provides the structural basis of DNA synthesis inhibition by brincidofovir and cidofovir, providing a rational strategy for new therapeutical development for mpox virus and other pox viruses.

Monkeypox: A re-emerging disease.

ABSTRACT: The virus known as monkeypox is the source of the zoonotic disease monkeypox, which was historically widespread in Central Africa and West Africa. The cases of monkeypox in humans are uncommon outside of West and Central Africa, but copious nonendemic nations outside of Africa have recently confirmed cases. People when interact with diseased animals, then, they may inadvertently contact monkeypox. There are two drugs in the market: brincidofovir and tecovirimat and both of these drugs are permitted for the cure of monkeypox by the US Food and Drug Administration. The present review summarizes the various parameters of monkeypox in context with transmission, signs and symptoms, histopathological and etiological changes, and possible treatment. Monkeypox is clinically similar to that of smallpox infection but epidemiologically, these two are different, the present study also signifies the main differences and similarities of monkeypox to that of other infectious diseases. As it is an emerging disease, it is important to know about the various factors related to monkeypox so as to control it on a very early stage of transmission.

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.

Brincidofovir for disease progression due to suspected tecovirimat resistance in association with advanced HIV.

A man with advanced HIV presented with verrucous plaques 2-3 months after initial mpox infection. He received two courses of tecovirimat without resolution of initial mpox lesions and development of new lesions raising concern for resistance. He was treated with two doses of brincidofovir and demonstrated improvement 6 months later.

The successful treatment of mpox with brincidofovir in renal transplant recipients-a report of 2 cases.

An mpox outbreak was declared in July 2022 by the world health organization (WHO). It causes a mild self-limiting disease however; in immunosuppressed hosts, it tends to cause severe disseminated infection. Most cases of mpox in sold organ transplant (SOT) recipients reported in the literature were treated with tecovirimat. Here we report two cases of severe disseminated mpox infection in renal transplant recipients that were successfully treated with brincidofovir. Both patients were discharged from the hospital with no immediate significant side effects from brincidofovir reported until the submission of this report.

Brincidofovir Effectively Inhibits Proliferation of Pseudorabies Virus by Disrupting Viral Replication.

Pseudorabies is an acute and febrile infectious disease caused by pseudorabies virus (PRV), a member of the family Herpesviridae. Currently, PRV is predominantly endemoepidemic and has caused significant economic losses among domestic pigs. Other animals have been proven to be susceptible to PRV, with a mortality rate of 100%. In addition, 30 human cases of PRV infection have been reported in China since 2017, and all patients have shown severe neurological symptoms and eventually died or developed various neurological sequelae. In these cases, broad-spectrum anti-herpesvirus drugs and integrated treatments were mostly applied. However, the inhibitory effect of the commonly used anti-herpesvirus drugs (e.g., acyclovir, etc.) against PRV were evaluated and found to be limited in this study. It is therefore urgent and important to develop drugs that are clinically effective against PRV infection. Here, we constructed a high-throughput method for screening antiviral drugs based on fluorescence-tagged PRV strains and multi-modal microplate readers that detect fluorescence intensity to account for virus proliferation. A total of 2104 small molecule drugs approved by the U.S. Food and Drug Administration (FDA) were studied and validated by applying this screening model, and 104 drugs providing more than 75% inhibition of fluorescence intensity were selected. Furthermore, 10 drugs that could significantly inhibit PRV proliferation in vitro were strictly identified based on their cytopathic effects, virus titer, and viral gene expression, etc. Based on the determined 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50), the selectivity index (SI) was calculated to be 26.3-3937.2 for these 10 drugs, indicating excellent drugability. The antiviral effects of the 10 drugs were then assessed in a mouse model. It was found that 10 mg/kg brincidofovir administered continuously for 5 days provided 100% protection in mice challenged with lethal doses of the human-origin PRV strain hSD-1/2019. Brincidofovir significantly attenuated symptoms and pathological changes in infected mice. Additionally, time-of-addition experiments confirmed that brincidofovir inhibited the proliferation of PRV mainly by interfering with the viral replication stage. Therefore, this study confirms that brincidofovir can significantly inhibit PRV both in vitro and in vivo and is expected to be an effective drug candidate for the clinical treatment of PRV infections.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.

5-Formylcytosine mediated DNA-peptide cross-link induces predominantly semi-targeted mutations in both Escherichia coli and human cells.

Histone proteins can become trapped on DNA in the presence of 5-formylcytosine (5fC) to form toxic DNA-protein conjugates. Their repair may involve proteolytic digestion resulting in DNA-peptide cross-links (DpCs). Here, we have investigated replication of a model DpC comprised of an 11-mer peptide (NH2-GGGKGLGK∗GGA) containing an oxy-lysine residue (K∗) conjugated to 5fC in DNA. Both CXG and CXT (where X = 5fC-DpC) sequence contexts were examined. Replication of both constructs gave low viability (<10%) in Escherichia coli, whereas TLS efficiency was high (72%) in HEK 293T cells. In E. coli, the DpC was bypassed largely error-free, inducing only 2 to 3% mutations, which increased to 4 to 5% with SOS. For both sequences, semi-targeted mutations were dominant, and for CXG, the predominant mutations were G→T and G→C at the 3'-base to the 5fC-DpC. In HEK 293T cells, 7 to 9% mutations occurred, and the dominant mutations were the semi-targeted G → T for CXG and T → G for CXT. These mutations were reduced drastically in cells deficient in hPol η, hPol ι or hPol ζ, suggesting a role of these TLS polymerases in mutagenic TLS. Steady-state kinetics studies using hPol η confirmed that this polymerase induces G → T and T → G transversions at the base immediately 3' to the DpC. This study reveals a unique replication pattern of 5fC-conjugated DpCs, which are bypassed largely error-free in both E. coli and human cells and induce mostly semi-targeted mutations at the 3' position to the lesion.

Evolved Readers of 5-Carboxylcytosine CpG Dyads Reveal a High Versatility of the Methyl-CpG-Binding Domain for Recognition of Noncanonical Epigenetic Marks.

Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5-methylation (mC), a total of four different 5-modifications can theoretically co-exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5-carboxylcytosine (caC) dyads based on the methyl-CpG-binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC-dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design.