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DNA, Volume 5, Issue 2 (June 2025) – 10 articles

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23 pages, 3859 KiB  
Article
Comparing the Efficiency of Different Methods for Reliable Results in Ancient DNA NGS Workflow
by Bence Kovács, Alexandra Gînguță, Petra Kiss, Kitti Maár, Oszkár Schütz, Gergely I. B. Varga and Endre Neparáczki
DNA 2025, 5(2), 26; https://doi.org/10.3390/dna5020026 - 19 May 2025
Viewed by 201
Abstract
Background/Objectives: Ancient DNA (aDNA) research workflows heavily depend on efficient aDNA extraction and NGS library preparation. In this study, we compared some of the commonly used laboratory protocols and compared the source of the bone material for sufficient and reliable results. Methods: We [...] Read more.
Background/Objectives: Ancient DNA (aDNA) research workflows heavily depend on efficient aDNA extraction and NGS library preparation. In this study, we compared some of the commonly used laboratory protocols and compared the source of the bone material for sufficient and reliable results. Methods: We executed a three-phase study. First, we analyzed about 2000 previously processed archaic bone samples and conducted a comparative analysis. The second phase involved a controlled experiment of five ancient individuals, with internal control, to further investigate the efficiency of some of the methods. In the third phase, we made a comparison between the efficiency of two enzymes used for library preparation. Results: Samples made from Pars petrosa resulted in the highest yield of endogenous DNA and longer fragment sizes compared to tooth or skeletal samples. DNA extraction made by MinElute columns preserved slightly longer fragments than the handmade silica suspension. NGS libraries indexed using AccuPrime Pfx produced slightly more consistent insert sizes compared to GoTaq G2. Samples prepared with GoTaq G2 contained slightly more unique molecules. The duplication rates showed no significant impact from enzyme choice. Conclusions: Pars petrosa remains the most reliable source of aDNA, with the extraction method using MinElute columns. While AccuPrime Pfx ensures precise NGS library preparation, a more economical choice of the GoTaq G2 enzyme is a viable alternative for degraded archaic samples. Full article
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9 pages, 1518 KiB  
Article
Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases
by Komal Chillar, Rohith Awasthy, Marina Tanasova and Shiyue Fang
DNA 2025, 5(2), 25; https://doi.org/10.3390/dna5020025 - 9 May 2025
Viewed by 218
Abstract
Background/Objective: Oligodeoxynucleotides (ODNs) containing base-labile modifications such as N4-acetyldeoxycytidine (4acC), N6-acetyladenosine (6acA), N2-acetylguanosine (2acG), and N4-methyoxycarbonyldeoxycytidine (4mcC) are highly challenging to synthesize because standard ODN synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, and there is a lack of [...] Read more.
Background/Objective: Oligodeoxynucleotides (ODNs) containing base-labile modifications such as N4-acetyldeoxycytidine (4acC), N6-acetyladenosine (6acA), N2-acetylguanosine (2acG), and N4-methyoxycarbonyldeoxycytidine (4mcC) are highly challenging to synthesize because standard ODN synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, and there is a lack of ideal alternative methods to solve the problem. The objective of this work is to explore the capability of the recently developed 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) method for the incorporation of multiple 4acC modifications into a single ODN molecule and the feasibility of using the method for the incorporation of the 6acA, 2acG and 4mcC modifications into ODNs. Methods: The sensitive ODNs were synthesized on an automated solid phase synthesizer using the Dmoc group as the linker and the methyl Dmoc (meDmoc) group for the protection of the exo-amino groups of nucleobases. Deprotection and cleavage were achieved under non-nucleophilic and weakly basic conditions. Results: The 4acC, 6acA, 2acG, and 4mcC were all found to be stable under the mild ODN deprotection and cleavage conditions. Up to four 4acC modifications were able to be incorporated into a single 19-mer ODN molecule. ODNs containing the 6acA, 2acG, and 4mcC modifications were also successfully synthesized. The ODNs were characterized using RP HPLC, capillary electrophoresis, gel electrophoresis and MALDI MS. Conclusions: Among the modified nucleotides, 4acC has been found in nature and proven beneficial to DNA duplex stability. A method for the synthesis of ODNs containing multiple 4acC modifications is expected to find applications in biological studies involving 4acC. Although 6acA, 2acG, and 4mcC have not been found in nature, a synthetic route to ODNs containing them is expected to facilitate projects aimed at studying their biophysical properties as well as their potential for antisense, RNAi, CRISPR, and mRNA therapeutic applications. Full article
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33 pages, 7606 KiB  
Review
DNA Replication in Time and Space: The Archaeal Dimension
by Anastasia Serdyuk and Thorsten Allers
DNA 2025, 5(2), 24; https://doi.org/10.3390/dna5020024 - 6 May 2025
Viewed by 336
Abstract
The ability of a nucleic acid molecule to self-replicate is the driving force behind the evolution of cellular life and the transition from RNA to DNA as the genetic material. Thus, the physicochemical properties of genome replication, such as the requirement for a [...] Read more.
The ability of a nucleic acid molecule to self-replicate is the driving force behind the evolution of cellular life and the transition from RNA to DNA as the genetic material. Thus, the physicochemical properties of genome replication, such as the requirement for a terminal hydroxyl group for de novo DNA synthesis, are conserved in all three domains of life: eukaryotes, bacteria, and archaea. Canonical DNA replication is initiated from specific chromosomal sequences termed origins. Early bacterial models of DNA replication proposed origins as regulatory points for spatiotemporal control, with replication factors acting on a single origin on the chromosome. In eukaryotes and archaea, however, replication initiation usually involves multiple origins, with complex spatiotemporal regulation in the former. An alternative replication initiation mechanism, recombination-dependent replication, is observed in every cellular domain (and viruses); DNA synthesis is initiated instead from the 3′ end of a recombination intermediate. In the domain archaea, species including Haloferax volcanii are not only capable of initiating DNA replication without origins but grow faster without them. This raises questions about the necessity and nature of origins. Why have archaea retained such an alternative DNA replication initiation mechanism? Might recombination-dependent replication be the ancestral mode of DNA synthesis that was used during evolution from the primordial RNA world? This review provides a historical overview of major advancements in the study of DNA replication, followed by a comparative analysis of replication initiation systems in the three domains of life. Our current knowledge of origin-dependent and recombination-dependent DNA replication in archaea is summarised. Full article
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26 pages, 6899 KiB  
Article
Unveiling the Genomic Basis of Antagonism and Plant Growth Promotion in the Novel Endophyte Bacillus velezensis Strain B.B.Sf.2
by Dimitra Douka, Tasos-Nektarios Spantidos, Panagiotis Katinakis and Anastasia Venieraki
DNA 2025, 5(2), 23; https://doi.org/10.3390/dna5020023 - 4 May 2025
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Abstract
Background/Objectives: The agriculture sector faces significant challenges due to global climate change, environmental stressors, and rapid population growth, compounded by unsustainable farming practices. This study investigates the potential of the endophytic bacterial strain B.B.Sf.2, isolated from the bark of Salvia fruticosa and identified [...] Read more.
Background/Objectives: The agriculture sector faces significant challenges due to global climate change, environmental stressors, and rapid population growth, compounded by unsustainable farming practices. This study investigates the potential of the endophytic bacterial strain B.B.Sf.2, isolated from the bark of Salvia fruticosa and identified as Bacillus velezensis through phylogenomic analyses. Methods: To address these issues, eco-friendly techniques, such as the application of plant-associated microbes, are gaining attention. Genome mining revealed numerous secondary metabolite biosynthetic gene clusters associated with plant growth promotion, biocontrol, colonization, and defense elicitation. Results: The strain exhibited strong antagonistic activity against phytopathogens, mediated by diffusible and volatile compound production, along with plant-growth-promoting traits and environmental adaptability. Genome mining revealed numerous secondary metabolite biosynthetic gene clusters associated with plant growth promotion, biocontrol, colonization, and defense elicitation. B.B.Sf.2 effectively inhibited Colletotrichum species causing olive anthracnose and suppressed Botrytis cinerea, the gray mold pathogen, in post-harvest studies on infected fruits. Bioautography of ethyl acetate extracts demonstrated bioactivity against B. cinerea, attributed to iturin-like metabolites. The extracts maintained bioactive properties regardless of fungal interaction. Furthermore, the strain significantly promoted the growth of Arabidopsis thaliana via diffusible and volatile compounds. Conclusions: Our results highlight the multifunctional potential of B.B.Sf.2 as a biocontrol and growth-promoting agent, warranting further evaluation in field applications to enhance sustainable agriculture. Full article
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26 pages, 964 KiB  
Review
Genomic and Epigenomic Plasticity in the Hypoxic Environment
by Emanuele Cricchi, Alessio Bertozzo, Martina Minisini and Claudio Brancolini
DNA 2025, 5(2), 22; https://doi.org/10.3390/dna5020022 - 4 May 2025
Viewed by 261
Abstract
Our genome has evolved a complex network of information designed to precisely regulate gene transcription. Commonly known as cis-regulatory elements, they represent those parts of DNA that are highly sensitive to environmental changes in the form of associated multi-protein complexes. Oxygen levels are [...] Read more.
Our genome has evolved a complex network of information designed to precisely regulate gene transcription. Commonly known as cis-regulatory elements, they represent those parts of DNA that are highly sensitive to environmental changes in the form of associated multi-protein complexes. Oxygen levels are an important environmental factor influencing a range of cellular activities, including cell survival. To respond to changes in oxygen levels, cells have developed an efficient and precise system for regulating gene expression. Cis-regulatory elements are the key hubs of this response and control the activation of the transcriptional response to hypoxia. In this review, we will discuss the complex genomic and epigenomic structures that are modulated by oxygen and control the activity of cis-regulatory elements and the adaptations to variations in O2 availability. Full article
(This article belongs to the Special Issue Epigenetics and Environmental Exposures)
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19 pages, 1387 KiB  
Review
Research Themes in KAT6A Syndrome: A Scoping Review
by Tanya Tripathi, Miya St John, Jordan Wright, Natacha Esber and David J. Amor
DNA 2025, 5(2), 21; https://doi.org/10.3390/dna5020021 - 27 Apr 2025
Viewed by 550
Abstract
Pathogenic variants in the KAT6A gene cause KAT6A syndrome, a neurodevelopmental disorder characterised by intellectual disability (ID), developmental delay, speech and language challenges, feeding difficulties, and skeletal abnormalities. This scoping review synthesises current knowledge on KAT6A syndrome, identifies key research themes, and supports [...] Read more.
Pathogenic variants in the KAT6A gene cause KAT6A syndrome, a neurodevelopmental disorder characterised by intellectual disability (ID), developmental delay, speech and language challenges, feeding difficulties, and skeletal abnormalities. This scoping review synthesises current knowledge on KAT6A syndrome, identifies key research themes, and supports the mission of advocacy groups like the KAT6 Foundation. A systematic search of five databases (Ovid MEDLINE, Ovid EMBASE, PubMed, Web of Science, and Scopus) was conducted from 1990 to 2024, including peer-reviewed articles, preprints, and conference abstracts published from 2022 onward. Of 771 citations retrieved, 111 full-text articles were reviewed, with 62 meeting the inclusion criteria. Data were synthesised into six themes: (1) the genotype and phenotype map, revealing a broad phenotypic spectrum with common features like ID, absent speech, and craniofacial dysmorphism, as well as rare features such as severe aplastic anaemia and pancraniosynostosis; (2) the neurodevelopmental profile, detailing communication deficits, sleep disturbances, and impaired adaptive functioning; (3) the epigenetic and developmental roles of KAT6A, highlighting its critical function in histone acetylation, chromatin remodelling, and gene regulation; (4) molecular biomarkers, identifying distinct DNA methylation episignatures and dysregulated cellular pathways; (5) drug discovery, with preliminary studies suggesting that pantothenate and L-carnitine may mitigate mitochondrial dysfunction and histone acetylation deficits, while RSPO2 overexpression reverses cognitive impairment in animal models; (6) phenotypic overlap with Rett syndrome and KAT6B-related disorders. This review underscores the complexity and variability of KAT6A syndrome, highlighting the need for multidisciplinary approaches to improving diagnosis, management, and development of therapies. Future research should focus on longitudinal studies, underrepresented phenotypes, biomarker identification, and robust therapeutic trials to enhance outcomes for affected individuals and their families. Full article
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14 pages, 1963 KiB  
Article
DNA Barcoding as a Tool for Surveying Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada
by Evgeny Ilyukhin and Svetlana Markovskaja
DNA 2025, 5(2), 20; https://doi.org/10.3390/dna5020020 - 21 Apr 2025
Viewed by 244
Abstract
Background/Objectives: Branch dieback and canker diseases caused by Cytospora species adversely impact the health of woody plants worldwide. Results: During this survey, 59 Cytospora isolates were obtained from symptomatic trees and shrubs growing in southwest Ontario and Saskatchewan, Canada. A DNA barcoding approach [...] Read more.
Background/Objectives: Branch dieback and canker diseases caused by Cytospora species adversely impact the health of woody plants worldwide. Results: During this survey, 59 Cytospora isolates were obtained from symptomatic trees and shrubs growing in southwest Ontario and Saskatchewan, Canada. A DNA barcoding approach combined with morphological characterization identified 15 known species of Cytospora associated with these diseases: C. chrysosperma, C. curvata, C. euonymina, C. hoffmannii, C. kantschavelii, C. leucosperma, C. leucostoma, C. nitschkeana, C. piceae, C. populina, C. pruinopsis, C. pruinosa, C. ribis, C. schulzeri, and C. sorbina. The most common species isolated from multiple hosts were C. sorbina (10), C. chrysosperma (8), C. nitschkeana (6), and C. pruinosa (6). A wide range of host associations, including non-conifer species, was observed for C. piceae. Conclusions: The obtained results contribute to the study of diversity, host affiliation, geographical distribution, and pathogenicity of Cytospora species occurring on woody plants in both natural habitats and agricultural systems. The findings support the effectiveness of using DNA barcodes in fungal taxonomy and plant pathology studies. Full article
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17 pages, 862 KiB  
Review
Chemical Versus Enzymatic Nucleic Acid Modifications and Genomic Stability
by Jonathan R. Cortez and Marie E. Migaud
DNA 2025, 5(2), 19; https://doi.org/10.3390/dna5020019 - 9 Apr 2025
Viewed by 524
Abstract
DNA damage and repair have been central themes in cellular biology research. Broadly, DNA damage is understood as modifications to canonical nucleotides that disrupt their function during transcription and replication. A deeper biochemical understanding of DNA damage is essential, as the genome governs [...] Read more.
DNA damage and repair have been central themes in cellular biology research. Broadly, DNA damage is understood as modifications to canonical nucleotides that disrupt their function during transcription and replication. A deeper biochemical understanding of DNA damage is essential, as the genome governs all cellular processes. We can classify DNA damage according to whether the modifications to the nucleic acid scaffold are chemically or enzymatically initiated. This distinction is important because chemical modifications are often irreversible, sometimes sparse, and difficult to detect or control spatially and replicate systematically. This can result in genomic damage or modifications to nucleotides in the nucleotide pool, which is less commonly studied. In contrast, enzymatic modifications are typically induced by the cell for specific purposes and are under strong regulatory control. Enzymatic DNA modifications also present a degree of sequence specificity and are often reversible. However, both types of DNA modifications contribute to cellular aging when poorly repaired and, as a result, remain incompletely understood. This review hopes to gather less studied mechanisms in nucleotide modifications and show research gaps in our current understanding of nucleotide biology. By examining the implications of these mechanisms on DNA modifications, in the nucleotide pool and genome, we may gain insights into innovative strategies for mitigating the effects of cellular aging. Full article
(This article belongs to the Special Issue Epigenetics and Environmental Exposures)
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10 pages, 952 KiB  
Article
Thyroid Hormone-Responsive Genes in Primary Cultures of Rat Hepatic Cells
by Nariaki Fujimoto and Shigeyuki Kitamura
DNA 2025, 5(2), 18; https://doi.org/10.3390/dna5020018 - 1 Apr 2025
Viewed by 322
Abstract
Background/Objectives: Thyroid hormones are key regulators in hepatic metabolic pathways. Although they regulate various hepatic genes, only a few are known to be under direct transcriptional regulation through thyroid hormone receptors. To better understand the roles of thyroid hormones in the liver, it [...] Read more.
Background/Objectives: Thyroid hormones are key regulators in hepatic metabolic pathways. Although they regulate various hepatic genes, only a few are known to be under direct transcriptional regulation through thyroid hormone receptors. To better understand the roles of thyroid hormones in the liver, it is critical to identify thyroid hormone-responsive genes at the cellular level. Methods: A cDNA microarray analysis was applied to primary cultures of rat hepatic cells treated with triiodothyronine (T3) at 10−9 M for 24 h to identify the differentially expressed genes. The identified gene expressions were further examined in vivo using F344 rats. The reporter gene assay was performed to investigate the transcriptional activity of the upstream region of the gene. Results: A limited number of genes were listed, and only three of them, pyridoxal kinase (Pdxk), phosphoenolpyruvate carboxykinase 1 (Pck1), and solute carrier family 17 member 2 (Slc17a2), were confirmed to be upregulated by quantitative RT-PCR. The mRNA expression of these genes increased in the livers of F344 rats after T3 injection, suggesting the physiological relevance in vivo. There are two partially conserved thyroid hormone-responsive elements (TREs) in the upstream region of the rat Pdxk gene. The reporter gene assay indicated that an imperfect TRE (5′-gGGTCAxxxxAGGaCt-3′) located at −2146 was sufficient for the thyroid hormone-induced transcription of the gene. Conclusions: The present study identified novel T3-responsive genes, pdxk and Slc17a2. Promoter analyses showed that a single TRE in the pdxk gene accounts for the transcriptional regulation by T3. Full article
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20 pages, 2014 KiB  
Review
Overview of Roles of Novel Components in the Regulation of DNA Damage Repair in BRCA1-Deficient Cancers: An Update
by Nhat Nguyen, Dominic Arris and Manh Tien Tran
DNA 2025, 5(2), 17; https://doi.org/10.3390/dna5020017 - 1 Apr 2025
Viewed by 487
Abstract
Cancers that arise from germline mutations of breast cancer associated gene 1 (BRCA1), which is a crucial player in homologous recombination (HR) DNA repair, are vulnerable to DNA-damaging agents such as platinum and PARP inhibitors (PARPis). Increasing evidence suggests that BRCA1 [...] Read more.
Cancers that arise from germline mutations of breast cancer associated gene 1 (BRCA1), which is a crucial player in homologous recombination (HR) DNA repair, are vulnerable to DNA-damaging agents such as platinum and PARP inhibitors (PARPis). Increasing evidence suggests that BRCA1 is an essential driver of all phases of the cell cycle, thereby maintaining orderly steps during cell cycle progression. Specifically, loss of BRCA1 activity causes the S-phase, G2/M, spindle checkpoints, and centrosome duplication to be dysregulated, thereby blocking cell proliferation and inducing apoptosis. In vertebrates, loss of HR genes such as BRCA1 and/or BRCA2 is lethal, since HR is a prerequisite for genome integrity. Thus, cancer cells utilize alternative DNA repair pathways such as non-homologous end joining (NHEJ) to cope with the loss of BRCA1 function. In this review, we attempt to update and discuss how these novel components are crucial for regulating DNA damage repair (DDR) in BRCA1-deficient cancers. Full article
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