DNA, Volume 5, Issue 3 (September 2025) – 10 articles

Background/Objectives: Circulating cell-free DNA (cfDNA) consists of genomic DNA (cf-nDNA) and mitochondrial DNA (cf-mtDNA) fragments released from cells primarily through apoptosis and necrosis. In healthy individuals, the main source of cfDNA is apoptosis, whereas in cancer patients, necrosis predominates. Considering that in vitro cfDNA models are valuable research tools, this study presents an in vitro characterization of cf-mtDNA patterns released into the culture medium by four human cell lines: normal dermal fibroblasts (Hs27), induced pluripotent stem cells (iPSCs), melanoma cells (BMel), and prostate cancer cells (PC3). Furthermore, as fetal bovine serum (FBS)—a widely used supplement in cell culture media—has been shown to contain bovine cfDNA, species-specific primers were employed to eliminate potential artifacts arising from this contamination in in vitro experiments. Methods: Fragmentation analysis of cf-mtDNA was conducted by amplifying the human MT-CYB gene and the D-loop region in four cell lines using species-specific primers. Two indices, Q and λ, were employed to quantify fragmentation. Results: These indices reveal that cancer cells exhibit the highest degree of fragmentation compared to fibroblasts, whereas stem cells show the lowest degree of fragmentation. This study identified species-specific primers for the human and bovine MT-CYB gene, confirming the presence of bovine cf-mtDNA in cell culture media supplemented with FBS. Conclusions: in vitro cellular models are useful for studying the mechanisms of cfDNA release and fragmentation; designed primers provide a reliable tool for assessing contamination across different growth time points minimizing interference errors and non-specific amplifications. Full article

Background/Objectives: Bolting in lettuce (Lactuca sativa L.) is highly sensitive to elevated temperatures, leading to premature flowering and reduced crop quality and yield. Although SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) is a well-known floral integrator in Arabidopsis, its role in heat-induced bolting in lettuce remains unclear. Methods: In this study, we generated CRISPR/Cas9-mediated LsSOC1 knockout (KO) lines and evaluated their phenotypes under high-temperature conditions. Results: LsSOC1-KO lines exhibited delayed bolting up to 18.6 days, and stem elongation was reduced by approximately 3.8 cm, which is equivalent to a 36.1% decrease compared to wild-type (WT) plants. Transcriptome analysis of leaf and bud tissues identified 32 up-regulated and 10 down-regulated genes common to leaf tissue (|log₂FC| ≥ 1, adjusted p < 0.05). Among them, GA20-oxidase1 was significantly down-regulated in both tissues, which may have contributed to delayed floral transition and possibly to reduced stem elongation, although tissue-specific regulation of gibberellin metabolism warrants further investigation. In contrast, genes encoding heat shock proteins, ROS-detoxification enzymes, and flavonoid biosynthetic enzymes were up-regulated, suggesting a dual role of LsSOC1 in modulating thermotolerance and floral transition. qRT-PCR validated the sustained suppression of flowering-related genes in LsSOC1 KO plants under 37 °C heat stress. Conclusions: These findings demonstrate that LsSOC1 is a key integrator of developmental and thermal cues, orchestrating both bolting and stress-responsive transcriptional programs. Importantly, delayed bolting may extend the harvest window and improve postharvest quality in lettuce, highlighting LsSOC1 as a promising genetic target for breeding heat-resilient leafy vegetables. Full article

Background/Objectives: Inteins are mobile genetic elements invading highly conserved genes across all domains of life and viruses. Five active intein insertion sites (MCM-a through e) had previously been identified and studied in the archaeal replicative helicase minichromosome maintenance (MCM) subunit gene mcm, making MCM an ideal system for dissecting the dynamics of multi-intein genes. However, work in this system thus far has been limited to particular archaeal groups. To better understand the dynamics and diversity of these inteins, MCM homologs spanning all archaeal groups were extracted from NCBI’s non-redundant protein sequence database, and the distribution and structural architectures of their inteins were characterized. Methods: The amino acid sequences of 4243 archaeal MCM homologs were retrieved from NCBI’s non-redundant protein sequence database. These sequences were systematically assessed for their intein content through within-group multiple sequence alignments. To characterize the inteins present at each site, extensive intein structure predictions and comparisons were performed. Phylogenetic analyses were used to investigate intein relatedness between and within sites, as well as the distribution of different MCM inteins in geographically overlapping populations of archaea. Results: In total, 11 active MCM intein insertion sites were identified, expanding on the previously known five. The insertion sites have varied invasion activity levels across archaeal groups, with Nanobdellati (DPANN) being the only group with all 11 sites active. In all but two (Methanonatronarchaeia and Hadarchaeota) of the archaeal groups studied where inteins were present, at least one MCM homolog was invaded by more than one intein. With respect to intein structure, within-intein insertions bearing semblance to DNA-binding domains were identified, with varied presence between inteins. Additionally, a study of archaeal MCM sequences of samples collected from the Atacama Desert in June 2013 revealed high MCM intein diversity levels. Conclusions: We identified six new active intein insertion sites in archaeal MCM, more than doubling the five previously known sites. All eleven intein insertion sites were either close to the ATP binding site, or the lined the channel through which the single-stranded DNA is pulled during the catalytic cycle of the helicase. Many of the analyzed inteins contained insertions bearing similarity to DNA-binding helix-turn-helix domains suggesting potential involvement in the intein homing process. Additionally, the high levels of MCM intein diversity observed in archaea from the Atacama Desert provide novel and strong support for a co-existence model of intein persistence. Full article

Background/Objectives: Male infertility is a major health concern with a complex etiopathology, yet a substantial proportion of cases remain idiopathic. Mitochondrial dysfunction and non-coding RNA (ncRNA) deregulation have both been implicated in impaired spermatogenesis, but their interplay remains poorly understood. This study aimed to identify infertility-specific variants in ncRNAs that affect mitochondrial dynamics and homeostasis and to explore their roles. Methods: Whole-genome sequencing (WGS) was performed on genomic DNA samples from teratozoospermic, asthenozoospermic, oligozoospermic, and normozoospermic men. Variants uniquely present in infertile individuals and mapped to ncRNAs that affect mitochondrial dynamics were selected and prioritized using bioinformatics tools. An independent transcriptomic validation was conducted using RNA-sequencing data from testicular biopsies of men with non-obstructive azoospermia (NOA) to determine whether the ncRNAs harboring WGS-derived variants were transcriptionally altered. Results: We identified several infertility-specific variants located in lncRNAs known to interact with mitochondrial regulators, including GAS5, HOTAIR, PVT1, MEG3, and CDKN2B-AS1. Transcriptomic analysis confirmed significant deregulation of these lncRNAs in azoospermic testicular samples. Bioinformatic analysis also implicated the disruption of lncRNA–miRNA–mitochondria networks, potentially contributing to mitochondrial membrane potential loss, elevated reactive oxygen species (ROS) production, impaired mitophagy, and germ cell apoptosis. Conclusions: Our integrative genomic and transcriptomic analysis highlights lncRNA–mitochondrial gene interactions as a novel regulatory layer in male infertility, while the identified lncRNAs hold promise as biomarkers and therapeutic targets. However, future functional studies are warranted to elucidate their mechanistic roles and potential for clinical translation in reproductive medicine. Full article

DNA connects the domains of genetic regulation and environmental interactions and plays a crucial role in neural development and cognitive function. The complex roles of genetic and epigenetic processes in brain development, synaptic plasticity, and higher-order cognitive abilities were reviewed in this study. Neural progenitors are formed and differentiated according to genetic instructions, whereas epigenetic changes, such as DNA methylation, dynamically control gene expression in response to external stimuli. These processes shape behavior and cognitive resilience by influencing neural identity, synaptic efficiency, and adaptation. This review also examines how DNA damage and repair mechanisms affect the integrity of neurons, which are essential for memory and learning. It also emphasizes how genetic predispositions and environmental factors interact to determine a person’s susceptibility to neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. Developments in gene-editing technologies, such as CRISPR, and non-viral delivery techniques provide encouraging treatment avenues for neurodegenerative disorders. This review highlights the fundamental role of DNA in coordinating the intricate interactions between molecular and environmental factors that underlie brain function and diseases. Full article

Background/Objectives: Temozolomide is an important drug used for the treatment of glioblastoma multiforme. Covalent conjugation of temozolomide to triplex-forming oligonucleotides could facilitate better sequence discrimination when targeted to DNA to lessen off-target effects and potentially reduce side-effects associated with conventional chemotherapy. The base sensitivity of temozolomide precludes use of basic deprotection conditions that typify the solid-supported synthesis of oligonucleotides. Methods: A novel di-iso-propylsilylene-linked solid support was developed and used in solid-supported synthesis of oligonucleotide conjugates. Results: Conditions were established whereby fully deprotected, solid-supported oligonucleotides could be prepared for derivatisation. Cleavage of the di-iso-propylsilylene linker was possible using mild, acidic conditions. Conclusions: The di-iso-propylsilylene-linked solid support was developed and shown to be compatible with base-sensitive oligonucleotide conjugate formation. The DNA triplex formation exhibited by a temozolomide oligonucleotide conjugate was equal in stability to the unconjugated control, opening new possibilities for sequence selective delivery of temozolomide to targeted DNA. Full article

Background: Medullary thyroid cancer (MTC), a neuroendocrine tumor originating from thyroid parafollicular C-cells, presents therapeutic challenges, particularly in advanced stages. While RET proto-oncogene mutations are known drivers, a comprehensive understanding of the broader somatic mutation landscape is needed to identify novel therapeutic targets and improve prognostication. This study leveraged the extensive AACR Project GENIE dataset to characterize MTC genomics. Methods: A retrospective analysis of MTC samples from GENIE examined recurrent somatic mutations, demographic/survival correlations, and copy number variations using targeted sequencing data (significance: p < 0.05). Results: Among 341 samples, RET mutations predominated (75.7%, mostly M918T), followed by HRAS (10.0%) and KRAS (5.6%), with mutual exclusivity between RET and RAS alterations. Recurrent mutations included KMT2D (5.3%), CDH11 (5.3%), ATM (5.0%), and TP53 (4.1%). NOTCH1 mutations were enriched in metastatic cases (p = 0.023). Preliminary associations included sex-linked mutations (BRAF/BRCA1/KIT in females, p = 0.028), and survival (ATM associated with longer survival, p = 0.016; BARD1/BLM/UBR5/MYH11 with shorter survival, p < 0.05), though limited subgroup sizes warrant caution. Conclusions: This large-scale genomic analysis confirms the centrality of RET and RAS pathway alterations in MTC and their mutual exclusivity. The association of NOTCH1 mutations with metastasis suggests a potential role in disease progression. While findings regarding demographic and survival correlations are preliminary, they generate hypotheses for future validation. This study enhances the genomic foundation for understanding MTC and underscores the need for integrated clinico-genomic datasets to refine therapeutic approaches. Full article

Background/Objectives: The potential of DNA as an information-dense storage medium has inspired a broad spectrum of creative systems. In particular, hybrid biomolecular systems that integrate new materials and chemistries with DNA could drive novel functions. In this work, we explore the potential for proteins to serve as molecular file addresses. We stored DNA-encoded data in yeast and leveraged yeast surface display to readily produce the protein addresses and make them easy to access on the cell surface. Methods: We generated yeast populations that each displayed a distinct protein on their cell surfaces. These proteins included binding partners for cognate antibodies as well as chromatin-associated proteins that bind post-translationally modified histone peptides. For each specific yeast population, we transformed a library of hundreds of DNA sequences collectively encoding a specific image file. Results: We first demonstrated that the yeast retained file-encoded DNA through multiple cell divisions without a noticeable skew in their distribution or a loss in file integrity. Second, we showed that the physical act of sorting yeast displaying a specific file address was able to recover the desired data without a loss in file fidelity. Finally, we showed that analog addresses can be achieved by using addresses that have overlapping binding specificities for target peptides. Conclusions: These results motivate further exploration into the advantages proteins may confer in molecular information storage. Full article

Microbial contamination is a critical challenge for the pharmaceutical industry, especially in thermosensitive sterile products, and can compromise their quality and safety. The accurate identification of microorganisms is essential to trace sources of contamination and adopt corrective measures. Although MALDI-TOF MS technology has revolutionized this process, its database limitations necessitate the use of complementary methods, such as sequencing 16S rRNA genes, housekeeping genes, and, in some cases, the entire genome. Advances in sequencing have expanded genomic taxonomy, increasing the accuracy of bacterial identification. The integration of these approaches significantly improves the reliability of identification, overcoming the limitations of isolated methods. Full article

In B-cell chronic lymphocytic leukemia (B-CLL), hypodiploidy is a rare but aggressive subtype of the disease with a very bad prognosis. Hypodiploidy, in contrast to normal B-CLL chromosomal aberrations, is marked by widespread genomic instability, which promotes treatment resistance and quick illness development. Its persistence after treatment implies that chromosomal loss gives cancerous clones a selection edge, which is made worse by telomere malfunction and epigenetic changes. Since thorough genetic profiling has a major impact on patient outcomes, advanced diagnostic methods are crucial for early detection. Treatment approaches must advance beyond accepted practices because of its resistance to traditional medicines. Hematopoietic stem cell transplantation (HSCT) and chimeric antigen receptor (CAR) T-cell therapy are two potential new therapeutic modalities. Relapse and treatment-related morbidity continue to be limiting concerns, despite the noteworthy improvements in outcomes in high-risk CLL patients receiving HSCT. Although more research is required, CAR T-cell treatment is effective in treating recurrent B-ALL and may also be used to treat B-CLL with hypodiploidy. Novel approaches are essential for enhancing patient outcomes and redefining therapeutic success when hypodiploidy challenges established treatment paradigms. Hypodiploidy is an uncommon yet aggressive form of B-CLL that has a very bad prognosis. Hypodiploidy represents significant chromosomal loss and structural imbalance, which contributes to a disordered genomic environment, in contrast to more prevalent cytogenetic changes. This instability promotes resistance to certain new drugs as well as chemoimmunotherapy and speeds up clonal evolution. Its persistence after treatment implies that hypodiploid clones have benefits in survival, which are probably strengthened by chromosomal segregation issues and damaged DNA repair pathways. Malignant progression and treatment failure are further exacerbated by telomere erosion and epigenetic dysregulation. The need for more sensitive molecular diagnostics is highlighted by the fact that standard karyotyping frequently overlooks hypodiploid clones, particularly those concealed by endoreduplication, despite the fact that these complications make early and correct diagnosis crucial. Hypodiploidy requires a move toward individualized treatment because of their link to high-risk genetic traits and resistance to conventional regimens. Although treatments like hematopoietic stem cell transplantation and CAR T-cells show promise, long-term management is still elusive. To improve long-term results and avoid early relapse, addressing this cytogenetic population necessitates combining high-resolution genomic technologies with changing therapy approaches. Full article