DNA-based Research Articles

Two-photon excitation two-dimensional fluorescence spectroscopy (2PE-2DFS) of the fluorescent nucleobase 6-MI.

Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength ~ 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength ~ 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.

DNA damage and repair in the nucleosome: insights from computational methods.

Cellular DNA is constantly exposed to endogenous or exogenous factors that can induce lesions. Several types of lesions have been described that can result from UV/ionizing irradiations, oxidative stress, or free radicals, among others. In order to overcome the deleterious effects of such damages, i.e., mutagenicity or cytotoxicity, cells possess a highly complex DNA repair machinery, involving repair enzymes targeting specific types of lesions through dedicated cellular pathways. In addition, DNA is highly compacted in the nucleus, the first level of compaction consisting of ~ 147 DNA base pairs wrapped around a core of histones, the so-called nucleosome core particle. In this complex environment, the DNA structure is highly constrained, and fine-tuned mechanisms involving remodeling processes are required to expose the DNA to repair enzymes and to facilitate the damage removal. However, these nucleosome-specific mechanisms remain poorly understood, and computational methods emerged only recently as powerful tools to investigate DNA damages in such complex systems as the nucleosome. In this mini-review, we summarize the latest advances brought out by computational approaches in the field, opening new exciting perspectives for the study of DNA damage and repair in the nucleosome context.

Mitochondrial transcription factor A (TFAM) has 5′-deoxyribose phosphate lyase activity in vitro

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5′-deoxyribose phosphate (5′dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase β, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5′dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5′dRp residues at a rate comparable to that of DNA polymerase (pol) β, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

Chair vs. Boat: Conformational Impacts on DNA Binding Capacity in Cu(II) Complexes Featuring cis‐1,4‐Cyclohexanedicarboxylate

AbstractTwo new coordination complexes were synthesized, which are formulated as [Cu2(cis‐1,4‐chdc)(4,4′‐Me2bpy)4] ⋅ 2(ClO4−) ⋅ H2O (1) and [Cu2(cis‐1,4‐chdc)(5,5′‐Me2bpy)4] ⋅ 2(ClO4−) ⋅ H2O (2), using cis‐1,4‐cyclohexanedicarboxylic acid (cis‐1,4‐H2chdc), 4,4′‐dimethyl‐2,2′‐bipyridine (4,4′‐Me2bpy), 5,5′‐dimethyl‐2,2′‐bipyridine (5,5′‐Me2bpy). Interestingly, cis‐1,4‐chdc adopts chair form in 1 and boat conformation in 2. This conformational change impacts the DNA binding capacities of the complexes as revealed by spectrophotometry, circular dichroism (CD) spectroscopy and docking studies. The complex 1 having the chair conformation shows higher affinity towards the DNA base pairs due to anti‐alignment of the planar aromatic pyridyl rings of 4,4′‐Me2bpy, which strongly intercalates with the adenosine base pairs of DNA by formation of three π–π stacking and two extra π‐positive stacking interactions between adenosine bases and positively charged nitrogen. Conversely, 5,5′‐Me2bpy with planar aromatic pyridyl rings of complex 2 shows bis‐intercalation with weak affinities toward base pairs by formation of two π–π stacking and one π‐positive stacking interactions.

Doppler ultrasonographic scan, gene expression and serum profile of immune, APPs and antioxidant markers in Egyptian buffalo–cows with clinical endometritis

The objective of this study was to elaborate Doppler ultrasonographic scan, genetic resistance and serum profile of markers associated with endometritis susceptibility in Egyptian buffalo–cows. The enrolled animals were designed as; twenty five apparently healthy buffalo–cows considered as a control group and twenty five infected buffalo with endometritis. There were significant (p < 0.05) increased of cervical diameter, endometrium thickness, uterine horn diameter, TAMEAN, TAMAX and blood flow through middle uterine artery with significant decrease of PI and RI values in endometritis buffalo–cows. Gene expression levels were considerably higher in endometritis-affected buffaloes than in resistant ones for the genes A2M, ADAMTS20, KCNT2, MAP3K4, MAPK14, FKBP5, FCAMR, TLR2, IRAK3, CCl2, EPHA4, and iNOS. The RXFP1, NDUFS5, TGF-β, SOD3, CAT, and GPX genes were expressed at substantially lower levels in endometritis-affected buffaloes. The PCR-DNA sequence verdicts of healthy and affected buffaloes revealed differences in the SNPs in the amplified DNA bases related to endometritis for the investigated genes. However, MAP3K4 elicited a monomorphic pattern. There was a significant decrease of red blood cells (RBCs) count, Hb and packed cell volume (PCV) with neutrophilia, lymphocytosis and monocytosis in endometritis group compared with healthy ones. The serum levels of Hp, SAA, Cp, IL-6, IL-10, TNF-α, NO and MDA were significantly (P˂0.05) increased, along with reduction of CAT, GPx, SOD and TAC in buffalo–cows with endometritis compared to healthy ones. The variability of Doppler ultrasonographic scan and studied genes alongside alterations in the serum profile of investigated markers could be a reference guide for limiting buffalo endometritis through selective breeding of natural resistant animals.

Generalized biomolecular modeling and design with RoseTTAFold All-Atom.

Deep-learning methods have revolutionized protein structure prediction and design but are presently limited to protein-only systems. We describe RoseTTAFold All-Atom (RFAA), which combines a residue-based representation of amino acids and DNA bases with an atomic representation of all other groups to model assemblies that contain proteins, nucleic acids, small molecules, metals, and covalent modifications, given their sequences and chemical structures. By fine-tuning on denoising tasks, we developed RFdiffusion All-Atom (RFdiffusionAA), which builds protein structures around small molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we designed and experimentally validated, through crystallography and binding measurements, proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and the light-harvesting molecule bilin.

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/μL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

Synthesis and Antioxidant Activities of Novel Pyrimidine Acrylamides as Inhibitors of Lipoxygenase: Molecular Modeling and In Silico Physicochemical Studies.

The pyrimidine ring is present in various biomolecules such as DNA and RNA bases, aminoacids, vitamins, etc. Additionally, many clinically used drugs including methotrexate and risperidone contain the pyrimidine heterocyclic scaffold as well. Pyrimidine derivatives present diverse biological activities including antioxidant and anticancer activities and can be considered as privileged scaffolds in drug discovery for the treatment of various diseases. Piperidine pyrimidine amides have gained significant attention due to their enzymatic inhibitory activity. Based on our experience and ongoing investigation on cinnamic acid derivatives, their hybrids and substituted pteridines acting as lipoxygenase inhibitors, antioxidants, anti-cancer, and anti-inflammatory agents a series of novel piperidine pyrimidine cinnamic acids amides have been designed and synthesized. The novel hybrids were studied for their antioxidant and anti-inflammatory potential. They exhibit moderate antioxidant activity in the DPPH assay which may be related to their bulkiness. Moreover, moderate to good lipid peroxidation inhibition potential was measured. With regards to their lipoxygenase inhibitory activity, however, two highly potent inhibitors out of the nine tested derivatives were identified, demonstrating IC50 values of 10.7 μM and 1.1 μM, respectively. Molecular docking studies to the target enzyme lipoxygenase support the experimental results.

Toxicity of persistent organic pollutants: a theoretical study

ContextPolychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two families of persistent organic pollutants that are dangerous as they remain in the atmosphere for long periods and are toxic for humans and animals. They are found all over the world, including the penguins of Antarctica. One of the mechanisms that explains the toxicity of these compounds is related to oxidative stress. The main idea of this theoretical research is to use conceptual density functional theory as a theory of chemical reactivity to analyze the oxidative stress that PCBs and PBDEs can produce. The electron transfer properties as well as the interaction with DNA nitrogenous bases of nine PCBs and ten PBDEs found in Antarctic penguins are investigated. From this study, it can be concluded that compounds with more chlorine or bromine atoms are more oxidizing and produce more oxidative stress. These molecules also interact directly with the nitrogenous bases of DNA, forming hydrogen bonds, and this may be an explanation for the toxicity. Since quinone-type metabolites of PCBs and PBDEs can cause neurotoxicity, examples of quinones are also investigated. Condensed Fukui functions are included to analyze local reactivity. These results are important as the reactivity of these compounds helps to explain the toxicity of PCBs and PBDEs.MethodsAll DFT computations were performed using Gaussian16 at M06-2x/6–311 + g(2d,p) level of theory without symmetry constraints. Electro-donating (ω-) and electro-accepting (ω +) powers were used as global response functions and condensed Fukui functions as local parameters of reactivity.

Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons.

Programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress premature termination codons (PTCs) that can lead to truncated and nonfunctional proteins. However, current methods to correct disease-associated PTCs suffer from low efficiency and limited precision. Here we develop RESTART v3, which uses near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold (range: 2.1- to 9.5-fold) higher editing efficiency than RESTART v2 in cultured cells and achieve functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense-to-nonsense codons, without affecting normal termination codons. Although off-target sites were detected, we did not observe changes to the coding information or the expression level of transcripts, and the overall natural tRNA abundance remained constant.

Breaking genetic shackles: The advance of base editing in genetic disorder treatment.

The rapid evolution of gene editing technology has markedly improved the outlook for treating genetic diseases. Base editing, recognized as an exceptionally precise genetic modification tool, is emerging as a focus in the realm of genetic disease therapy. We provide a comprehensive overview of the fundamental principles and delivery methods of cytosine base editors (CBE), adenine base editors (ABE), and RNA base editors, with a particular focus on their applications and recent research advances in the treatment of genetic diseases. We have also explored the potential challenges faced by base editing technology in treatment, including aspects such as targeting specificity, safety, and efficacy, and have enumerated a series of possible solutions to propel the clinical translation of base editing technology. In conclusion, this article not only underscores the present state of base editing technology but also envisions its tremendous potential in the future, providing a novel perspective on the treatment of genetic diseases. It underscores the vast potential of base editing technology in the realm of genetic medicine, providing support for the progression of gene medicine and the development of innovative approaches to genetic disease therapy.

Enhancement of intrinsic guanine fluorescence by protonation in DNA of various structures

Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides.

RNA base editors: The emerging approach of RNA therapeutics.

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m6A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.

Significant downregulation of Alzheimer's amyloid-β levels enabled by engineered DNA nanomaterials

Although there are no effective therapies to block or reverse Alzheimer's disease (AD) progression at present, a promising therapeutic strategy is to reduce levels of amyloid-β (Aβ) proteins, which drive the formation of amyloid plaque, a primary hallmark in AD brains. Herein, we report that amphiphilic lipid-DNA molecules (LD) were designed by incorporating a long alkyl chain into the nucleotide base. It significantly down-regulated Alzheimer's Aβ levels in vivo and in vitro. In contrast to small-molecule chemical drugs and antibody therapies, the assembled DNA nanoparticles allowed them to effectively cross the blood-brain barrier (BBB) and accumulate in the brain, increasing the therapeutic effects. Notably, lipid-DNA downregulated the levels of Aβ peptides significantly in vitro. AD mice model experiments demonstrated that the LD-treated groups exhibited a rapid cognition behavioral improvement, which was associated with brain engagement of LD and reduced Aβ levels. Thus, the molecularly engineered DNA nanomaterials effectively regulated Aβ peptides. This work might provide a promising DNA engineering strategy for AD treatment.

IR and Raman markers of the interactions between MoS2 and pyrimidine bases

Changes in vibrational spectra are among the most important manifestations of the interaction between transition metal dichalcogenides and nucleic acid bases (NAB). Infrared and Raman spectra were calculated using the DFT/M06-2X method for the most stable stacked and covalently bonded complexes of pyrimidine bases with MoS2. Obtained spectra were analyzed to determine the spectral markers of the interactions between the pyrimidine bases and MoS2. We found that interaction with MoS2 leads to significant changes in both frequencies and intensities of NAB vibrations. The correlation between the changes and interaction energies of the NAB molecules with MoS2 was demonstrated. In general, changes in the vibrational frequencies in complexes with covalent bonds are significantly greater than ones in stacked ones. For the bonded complexes, the most significant changes are observed for those fragments of the NAB molecules that are directly involved in the formation of the covalent bonds. Calculations predict the decrease of the IR intensity and Raman activities of the NAB molecules in the spectra of stacked complexes and their increase in the spectra of covalently bonded complexes.

Differential effects of benzo[a]pyrene exposure on glutathione and purine metabolism in keratinocytes: Dose-dependent and UV co-exposure effects.

Polycyclic aromatic hydrocarbons with the key substance benzo[a]pyrene (B[a]P) are widespread pollutants in the environment and at working places. Nonetheless, the exact underlying mechanisms of toxicological effects caused by B[a]P especially in absence and presence of UV irradiation remain uncertain. This study examines variations in exposure conditions: low B[a]P (4 nM), low B[a]P + UV and high B[a]P (4 μM), selected based on pertinent cytotoxicity assessments. Following cell viability evaluations post-treatment with varied B[a]P concentrations and UV irradiation, the identified concentrations underwent detailed metabolomic analysis via gas chromatography-mass spectrometry. Subsequently, resulting changes in metabolic profiles across these distinct exposure groups are comprehensively compared. Chemometric analyses showed modest regulation of metabolites after low B[a]P exposure compared to control conditions. High B[a]P and low B[a]P + UV exposure significantly increased regulation of metabolic pathways, indicating that additional UV irradiation plus low B[a]P is as demanding for the cells as higher B[a]P treatment alone. Further analysis revealed exposure-dependent regulation of glutathione-important for oxidative defence-and purine metabolism-important for DNA base synthesis. Only after low B[a]P, oxidative defence appeared to be able to compensate for B[a]P-induced perturbations of the oxidative homeostasis. In contrast, purine metabolism already responded towards adversity at low B[a]P. The metabolomic results give an insight into the mechanisms leading to the toxic response and confirm the strong effects of co-exposure on oxidative defence and DNA repair in the model studied.

Regulatory activity is the default DNA state in eukaryotes.

Genomes encode for genes and non-coding DNA, both capable of transcriptional activity. However, unlike canonical genes, many transcripts from non-coding DNA have limited evidence of conservation or function. Here, to determine how much biological noise is expected from non-genic sequences, we quantify the regulatory activity of evolutionarily naive DNA using RNA-seq in yeast and computational predictions in humans. In yeast, more than 99% of naive DNA bases were transcribed. Unlike the evolved transcriptome, naive transcripts frequently overlapped with opposite sense transcripts, suggesting selection favored coherent gene structures in the yeast genome. In humans, regulation-associated chromatin activity is predicted to be common in naive dinucleotide-content-matched randomized DNA. Here, naive and evolved DNA have similar co-occurrence and cell-type specificity of chromatin marks, challenging these as indicators of selection. However, in both yeast and humans, extreme high activities were rare in naive DNA, suggesting they result from selection. Overall, basal regulatory activity seems to be the default, which selection can hone to evolve a function or, if detrimental, repress.

Programmable RNA base editing via targeted modifications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editors are powerful tools in biology and hold great promise for the treatment of human diseases. Advanced DNA base editing tools, such as cytosine base editor and adenine base editor, have been developed to correct permanent mistakes in genetic material. However, undesired off-target edits would also be permanent, which poses a considerable risk for therapeutics. Alternatively, base editing at the RNA level is capable of correcting disease-causing mutations but does not lead to lasting genotoxic effects. RNA base editors offer temporary and reversible therapies and have been catching on in recent years. Here, we summarize some emerging RNA editors based on A-to-inosine, C-to-U and U-to-pseudouridine changes. We review the programmable RNA-targeting systems as well as modification enzyme-based effector proteins and highlight recent technological breakthroughs. Finally, we compare editing tools, discuss limitations and opportunities, and provide insights for the future directions of RNA base editing.

DNA and RNA base editors can correct the majority of pathogenic single nucleotide variants

The majority of human genetic diseases are caused by single nucleotide variants (SNVs) in the genome sequence. Excitingly, new genomic techniques known as base editing have opened efficient pathways to correct erroneous nucleotides. Due to reliance on deaminases, which have the capability to convert A to I(G) and C to U, the direct applicability of base editing might seem constrained in terms of the range of mutations that can be reverted. In this evaluation, we assess the potential of DNA and RNA base editing methods for treating human genetic diseases. Our findings indicate that 62% of pathogenic SNVs found within genes can be amended by base editing; 30% are G>A and T>C SNVs that can be corrected by DNA base editing, and most of them by RNA base editing as well, and 29% are C>T and A>G SNVs that can be corrected by DNA base editing directed to the complementary strand. For each, we also present several factors that affect applicability such as bystander and off-target occurrences. For cases where editing the mismatched nucleotide is not feasible, we introduce an approach that calculates the optimal substitution of the deleterious amino acid with a new amino acid, further expanding the scope of applicability. As personalized therapy is rapidly advancing, our demonstration that most SNVs can be treated by base editing is of high importance. The data provided will serve as a comprehensive resource for those seeking to design therapeutic base editors and study their potential in curing genetic diseases.

No evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 nanoparticles in three mammalian cell lines despite the initial reduction of cellular mitochondrial activity

Abstract We studied the effects of Aeroxide P25 titanium dioxide nanoparticles (TiO2 NPs) with a diameter of 21 nm on induction of DNA damage and long-term survival of three human cell lines: hepatocellular liver carcinoma HepG2, colorectal adenocarcinoma HT29 and lung carcinoma A549. The endpoints examined were DNA breakage estimated by the comet assay and oxidative base damage recognized by formamide-pyrimidine glycosylase (FPG) estimated with the FPG+ comet assay, frequencies of histone γH2AX foci and micronuclei, apoptosis, cell metabolic activity measured by mitochondrial activity (MTT) assay and long-term survival measured by colony-forming ability. Each cell line had a different pattern of DNA breakage and base damage vs. nanoparticle (NP) concentration and treatment time. There was no increase in the frequencies of histone γH2AX foci and micronuclei as compared to those in the untreated cells. In parallel with these results, no induction of apoptosis has been found in none of the cell lines tested. The reported experiments provided no evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 NPs, despite a slight decrease in mitochondrial activity and cell survival during the first 72 h.