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.

Nanopore-Based Single-Molecule Investigation of Cation Effect on the i-Motif Structure.

The i-motif, a secondary structure of a four-helix formed by cytosine-rich DNA (i-DNA) through C-C+ base pairing, is prevalent in human telomeres and promoters. This structure creates steric hindrance, thereby inhibiting both gene expression and protein coding. The conformation of i-DNA is intricately linked to the intracellular ionic environment. Hence, investigating its conformation under various ion conditions holds significant importance. In this study, we explored the impact of cations on the i-motif structure at the single-molecule level using the α-hemolysin (α-HL) nanochannel. Our findings reveal that the ability of i-DNA to fold into the i-motif structure follows the order Cs+ > Na+ > K+ > Li+ for monovalent cations. Furthermore, we observed the interconversion of single-stranded DNA (ss-DNA) and the i-motif structure at high and low concentrations of Mg2+ and Ba2+ electrolyte solutions. This study not only has the potential to extend the application of i-motif-based sensors in complex solution environments but also provides a new idea for the detection of metal ions.

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.

Using a hybrid neural network architecture for DNA sequence representation: A study on N4-methylcytosine sites.

N4-methylcytosine (4mC) is a modified form of cytosine found in DNA, contributing to epigenetic regulation. It exists in various genomes, including the Rosaceae family encompassing significant fruit crops like apples, cherries, and roses. Previous investigations have examined the distribution and functional implications of 4mC sites within the Rosaceae genome, focusing on their potential roles in gene expression regulation, environmental adaptation, and evolution. This research aims to improve the accuracy of predicting 4mC sites within the genome of Fragaria vesca, a Rosaceae plant species. Building upon the original 4mc-w2vec method, which combines word embedding processing and a convolutional neural network (CNN), we have incorporated additional feature encoding techniques and leveraged pre-trained natural language processing (NLP) models with different deep learning architectures including different forms of CNN, recurrent neural networks (RNN) and long short-term memory (LSTM). Our assessments have shown that the best model is derived from a CNN model using fastText encoding. This model demonstrates enhanced performance, achieving a sensitivity of 0.909, specificity of 0.77, and accuracy of 0.879 on an independent dataset. Furthermore, our model surpasses previously published works on the same dataset, thus showcasing its superior predictive capabilities.

Comprehensive analysis of the editing window of C-to-T TALE base editors.

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.

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.

Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

The front cover artwork is provided by Prof. Papadantonakis' group. The image shows a Watson-Crick Guanine-Cytosine pair, and the difference between vertical and adiabatic ionization potentials. Read the full text of the Research Article at 10.1002/cphc.202300946.

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.

A DFTB study on the electronic response of encapsulated DNA nucleobases onto chiral CNTs as a sequencer.

Sequencing the DNA nucleobases is essential in the diagnosis and treatment of many diseases related to human genes. In this article, the encapsulation of DNA nucleobases with some of the important synthesized chiral (7, 6), (8, 6), and (10, 8) carbon nanotubes were investigated. The structures were modeled by applying density functional theory based on tight binding method (DFTB) by considering semi-empirical basis sets. Encapsulating DNA nucleobases on the inside of CNTs caused changes in the electronic properties of the selected chiral CNTs. The results confirmed that van der Waals (vdW) interactions, π-orbitals interactions, non-bonded electron pairs, and the presence of high electronegative atoms are the key factors for these changes. The result of electronic parameters showed that among the CNTs, CNT (8, 6) is a suitable choice in sequencing guanine (G) and cytosine (C) DNA nucleobases. However, they are not able to sequence adenine (A) and thymine (T). According to the band gap energy engineering approach and absorption energy, the presence of G and C DNA nucleobases decreased the band gap energy of CNTs. Hence selected CNTs suggested as biosensor substrates for sequencing G and C DNA nucleobases.

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.

Excited States in Single-Stranded and i-Motif DNA with Silver Ions.

We have studied the excited states and structural properties for the complexes of cytosine (dC)10 chains with silver ions (Ag+) in a wide range of the Ag+ to DNA ratio (r) and pH conditions using circular dichroism, steady-state absorption, and fluorescence spectroscopy along with the ultrafast fluorescence upconversion technique. We also calculated vertical electronic transition energies and determined the nature of the corresponding excited states in some models of the cytosine-Ag+ complexes. We show that (dC)10 chains in the presence of silver ions form a duplex stabilized by C-Ag+-C bonds. It is also shown that the i-motif structure formed by (dC)10 chains is destabilized in the presence of Ag+ ions. The excited-state properties in the studied complexes depend on the amount of binding ions and the binding sites, which is supported by the calculations. In particular, new low-lying excited states appear when the second Ag+ ion interacts with the O atom of cytosine in the C-Ag+-C pairs. A similar picture is observed in the case when one Ag+ ion interacts with one cytosine via the N7 atom.

Chemical Repair of Radical Damage to the GC Base Pair by DNA-Bound Bisbenzimidazoles.

The migration of an electron-loss center (hole) in calf thymus DNA to bisbenzimidazole ligands bound in the minor groove is followed by pulse radiolysis combined with time-resolved spectrophotometry. The initially observed absorption spectrum upon oxidation of DNA by the selenite radical is consistent with spin on cytosine (C), as the GC• pair neutral radical, followed by the spectra of oxidized ligands. The rate of oxidation of bound ligands increased with an increase in the ratio (r) ligands per base pair from 0.005 to 0.04. Both the rate of ligand oxidation and the estimated range of hole transfer (up to 30 DNA base pairs) decrease with the decrease in one-electron reduction potential between the GC• pair neutral radical of ca. 1.54 V and that of the ligand radicals (E0', 0.90-0.99 V). Linear plots of log of the rate of hole transfer versus r give a common intercept at r = 0 and a free energy change of 12.2 ± 0.3 kcal mol-1, ascribed to the GC• pair neutral radical undergoing a structural change, which is in competition to the observed hole transfer along DNA. The rate of hole transfer to the ligands at distance, R, from the GC• pair radical, k2, is described by the relationship k2 = k0 exp(constant/R), where k0 includes the rate constant for surmounting a small barrier.

Plasmon-emitter coupling in cytosine-rich hairpin DNA-templated silver nanoclusters: Thermal reversibility, white light emission, and dynamics inside live cells.

Bio-templated luminescent noble metal nanoclusters (NCs) have attracted great attention for their intriguing physicochemical properties. Continuous efforts are being made to prepare NCs with high fluorescence quantum yield (QY), good biocompatibility, and tunable emission properties for their widespread practical applications as new-generation environment-friendly photoluminescent materials in materials chemistry and biological systems. Herein, we explored the unique photophysical properties of silver nanoclusters (AgNCs) templated by cytosine-rich customized hairpin DNA. Our results indicate that a 36-nucleotide containing hairpin DNA with 20 cytosine (C20) in the loop can encapsulate photostable red-emitting AgNCs with an absolute QY of ∼24%. The luminescent properties in these DNA-templated AgNCs were found to be linked to the coupling between the surface plasmon and the emitter. These AgNCs exhibited excellent thermal sensitivity and were employed to produce high-quality white light emission with an impressive color rendering index of 90 in the presence of dansyl chloride. In addition, the as-prepared luminescent AgNCs possessing excellent biocompatibility can effectively mark the nuclear region of HeLa cells and can be employed as a luminescent probe to monitor the cellular dynamics at a single molecular resolution.

The bisulfite reaction with cytosine and genomic DNA structure.

The bisulfite reaction with native DNA has been extensively employed in the detection of non-B DNA structures that can form spontaneously in DNA. These sequences are dynamic in that they can adopt both normal Watson-Crick paired B-DNA or unusual structures like the Triplex, G-Quadruplex, i-motif and Cruciform or Hairpin. Considerable evidence now suggests that these dynamic sequences play roles in both epigenetics and mutagenesis. The bisulfite reaction with native DNA offers a key approach to their detection. In this application whole cells, isolated nuclei or isolated DNA are treated with bisulfite under non-denaturing conditions in order to detect bisulfite accessible regions DNA that are associated with these structures. Here I review the stereochemistry of the bisulfite reaction, the electronic structure of its DNA cytosine substrates and its application in the detection of unusual structures in native DNA.

Utility of intercalator displacement assays for screening of ligands for i-motif DNA structures.

Cytosine rich sequences can form intercalated, i-motif DNA structures stabilized by hemi-protonated cytosine:cytosine base pairing. These sequences are often located in regulatory regions of genes such as promoters. Ligands targeting i-motif structures may provide potential leads for treatments for genetic disease. The focus on ligands interacting with i-motif DNA has been increasing in recent years. Here, we describe the fluorescent intercalator displacement (FID) assay using thiazole orange binding i-motif DNA and assess the binding affinity of a ligand to the i-motif DNA by displacing thiazole orange. This provides a time and cost-effective high throughput screening of ligands against secondary DNA structures for hit identification.

Potentiometric titrations to study ligand interactions with DNA i-motifs.

i-Motifs are non-canonical secondary structures of DNA formed by mutual intercalation of hemi-protonated cytosine-cytosine base pairs, most typically in slightly acidic conditions (pH<7.0). These structures are well-studied in vitro and have recently been suggested to exist in cells. Despite nearly a decade of active research, the quest for small-molecule ligands that could selectively bind to and stabilize i-motifs continues, and no reference, bona fide i-motif ligand is currently available. This is, at least in part, due to the lack of robust methods to assess the interaction of ligands with i-motifs, since many techniques well-established for studies of other secondary structures (such as CD-, UV-, and FRET-melting) may generate artifacts when applied to i-motifs. Here, we describe an implementation of automated, potentiometric (pH) titrations as a robust isothermal method to assess the impact of ligands or cosolutes on thermodynamic stability of i-motifs. This approach is validated through the use of a cosolute previously known to stabilize i-motifs (PEG2000) and three small-molecule ligands that are able to stabilize, destabilize, or have no effect on the stability of i-motifs, respectively.

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.

Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

Gas-phase and aqueous vertical ionization potentials, vIPgas and vIPaq respectively and measurements of the molecular electrostatic and local ionization maps calculated at the DFT/B3LYP-D3/ 6-311+G** level of theory and the C-PCM reaction field model for single- and double-stranded CpG and 5MeCpG pairs show that the vIPaq for single- and double-stranded pairs of C-G and 5MeC-G are practically the same, in the range of 5.79 to 5.81 eV. The aqueous adiabatic ionization potentials for single-stranded CpG and 5MeCpG are 5.52 eV and 5.51 eV respectively and they reflect the nuclear reorganization that takes place after the abstraction of the electron. The aqueous adiabatic ionization energy values that correspond to the CpG+. radical cation and the hydrated electron, e-,, being at infinite distance, adIPaq+Vo, are 3.92 eV and 3.91 eV respectively with (Vo=-1.6 eV) Analysis of data suggest that the HOMO-LUMO energy gap in the hard/soft-acid/base (HSAB) concept cannot be used a priori to determine the effect of cytosine methylation on the guanine enhanced oxidative damage in DNA.

Prediction of DNA i-motifs via machine learning.

i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine.

Selective Formation of Pd-DNA Hybrids Using Tailored Palladium-Mediated Base Pairs: Towards Heteroleptic Pd-DNA Systems.

The formation of highly organized metal-DNA structures has significant implications in bioinorganic chemistry, molecular biology and material science due to their unique properties and potential applications. In this study, we report on the conversion of single-stranded polydeoxycytidine (dC15 ) into a Pd-DNA supramolecular structure using the [Pd(Aqa)] complex (Aqa=8-amino-4-hydroxyquinoline-2-carboxylic acid) through a self-assembly process. The resulting Pd-DNA assembly closely resembles a natural double helix, with continuous [Pd(Aqa)(C)] (C=cytosine) units serving as palladium-mediated base pairs, forming interbase hydrogen bonds and intrastrand stacking interactions. Notably, the design of the [Pd(Aqa)] complex favours the interaction with cytosine, distinguishing it from our previously reported [Pd(Cheld)] complex (Cheld=chelidamic acid). This finding opens possibilities for creating heteroleptic Pd-DNA hybrids where different complexes specifically bind to nucleobases. We confirmed the Pd-DNA supramolecular structural assembly and selective binding of the complexes using NMR spectroscopy, circular dichroism, mass spectrometry, isothermal titration calorimetry, and DFT calculations.