Search Results (189)

The reusability of enzymes is a fundamental aspect of sustainable biotechnology and the development of biosensors. This study presents one of the first quantitative evaluations of DNA polymerase reusability by utilizing integrated quartz crystal microbalance (QCM) kinetics and real-time monitoring of exonuclease activity. The results showed that immobilized T7 DNA polymerase retained approximately 50% of its initial activity after three 90-min cycles and around 20% after five cycles. Significantly lower activities were observed for shorter, 45-min cycles. This indicates an unexpected time-dependent enhancement in stability for longer reaction times. The findings suggest a promising trend in enzyme stability and reusability, establishing a quantitative relationship between reaction duration and enzyme performance. This relationship offers a scalable pathway for the regeneration of biosensors and for sustainable enzymatic catalysis. Additionally, the work provides a transferable framework that can be applied to other DNA-processing enzymes, which supports long-term biosensor performance and industrial biocatalysis. The demonstrated approach offers a transferable and scalable methodology for the development of reusable polymerase-based biosensors and sustainable biocatalytic systems. Full article

Isothermal titration calorimetry (ITC) provides direct insight into the energetics of DNA polymerase function, including binding, catalysis, and exonuclease activity. We characterized a Phi29 mutant polymerase (SS_01) engineered to incorporate non-natural nucleotides in the presence of Mg²⁺, a function absent in the wild-type enzyme. ITC analyses revealed that SS_01 binding to the primed template was strongly influenced by metal ions. In the presence of Mg²⁺, the polymerase displayed tight binding (KD = 243 nM) and a clear exothermic signal, indicating activation of a large fraction of catalytically competent molecules. By contrast, in the presence of Ca²⁺, binding produced weaker exothermic signals (KD = 317 nM), suggesting less efficient binding complex formation. During dNTP- or oligonucleotide-tagged dNTP-driven polymerization, ITC profiles with Mg²⁺ exhibited pronounced endothermic heat changes, whereas with Ca²⁺, only minimal heat changes were observed. When binding only oligonucleotide-tagged dNTPs, the polymerases showed distinct thermodynamic behavior: in the presence of Mg²⁺, high substrate concentrations induced endothermic responses, while in the absence of catalytic ions, binding remained exothermic. Exonuclease activity monitored using unmodified oligonucleotides yielded strong exothermic signals in the presence of Mg²⁺ but weak responses in the presence of Ca²⁺, confirming strict ion dependence. Together, these data demonstrate that ITC directly captures the metal ion-dependent energetics of SS_01, providing mechanistic insight into its polymerization and exonuclease functions. Full article

Transcription–replication conflicts (TRCs) often occur in cells and cause DNA replication fork stalling. In this study, we investigated the interplay of RNA polymerase (RNAP), DNA polymerase, and RNase H1 (RH1) during head-on TRC in vitro with precise control over the reaction conditions. We show that it is a catalytically competent transcription elongation complex (TEC) that interferes with the action of both the Klenow fragment and full-length DNA Pol I. An incompetent RNAP complex with an R-loop stimulates the 3′→5′ exonuclease activity and pauses the DNA polymerase during head-on TRC. As RNAP advances along the DNA template, elongating the RNA, the head-on TRC is slowly overcome in our model system, likely through the reassociation of the displaced DNA polymerase with the nontemplate DNA strand upstream of RNAP. An isolated R-loop containing an 11-nt heteroduplex (R-loop-11) does not interfere with DNA replication by the Klenow fragment. For DNA Pol I, such an R-loop also does not stall replication but stimulates its 3′→5′ exonuclease activity. We demonstrate that a stalled Klenow fragment does not interfere with transcription, whereas a Klenow fragment moving along the TRC substrate towards RNAP alters the kinetics of RNAP. Stalled DNA Pol I does not stop RNAP but stimulates its endonuclease activity. We find that RH1 alone does not displace stalled RNAP from a competent TEC containing R-loop-11 and does not resolve the head-on TRC. On the other hand, RH1 displaces RNAP from the incompetent complex with the TRC substrate. This eliminates the stimulation of the 3′→5′ exonuclease activity of DNA polymerase during head-on TRC. Full article

This paper does not describe the results of a systematic search for the mechanism of action of cyclophosphamide and the consequences and possible indications arising from this mechanism. Rather, it describes a puzzle in which our own results, with some of them being very old, were re-evaluated with the latest biochemical knowledge and supplemented by results from the scientific literature. The mechanism of action of cyclophosphamide, which has been indispensable in clinical practice for 60 years, was unknown until recently simply because biochemical knowledge was lacking and because results from in vitro experiments were uncritically extrapolated to in vivo conditions. In vitro, the DNA alkylating metabolite phosphoramide mustard (PAM) is formed from the CP metabolite aldophosphamide (ALD) by phosphate and bicarbonate ion-catalyzed β-elimination of acrolein; in vivo, however, ALD is cleaved by phosphoesterases or DNA polymerase δ and ε, which are associated with 3′-5′ exonucleases, into the complementary metabolites PAM and 3-hydroxypropanal (HPA). The following describes the mechanism of action of CP, namely the complementary interaction of alkylating PAM and apoptosis-enhancing HPA, and it is shown that by optimizing the complementary effects of PAM and HPA, the antitumor efficacy in the P388 mouse tumor model can be increased by more than ten thousand-fold. Further experiments show that by optimizing the interaction of DNA alkylation and enhancing the resulting apoptosis by HPA, the formation of resistant metastases can be prevented and low-toxicity chemotherapy can be achieved. Full article

MicroRNAs (miRNAs) are key regulators of gene expression with critical roles in oncogenic signaling. Endometrial cancer (EC) has been redefined with the identification of POLE-ultramutated tumors which, despite their hypermutated phenotype, show more favorable prognosis. We profiled miRNA expression in tumor tissues from forty (40) EC patients and twenty (20) healthy controls using qPCR panels. POLE exonuclease domain mutations (P286R, V411L) were genotyped, and subgroup analyses were conducted between POLE-mutated (n = 7) and POLE-wild-type (n = 33) tumors. Bioinformatic analyses included validated miRNA–mRNA interactions, target enrichment, and Gene Ontology (GO) pathway mapping. Comparison of EC versus healthy endometrium revealed 50 significantly dysregulated (∣log₂ (FoldReg)∣ > 1 and BH FDR < 0.05) miRNAs, including up-regulation of the oncogenic hsa-miR-181a-5p, hsa-miR-23a-3p, hsa-miR-200c-3p, and down-regulation of tumor-suppressive let-7 family members. Target enrichment implicated canonical oncogenic regulators such as MYC, TP53, and VEGFA. POLE-mutated tumor analysis demonstrated a miRNA signature, with 19 miRNAs significantly down-regulated, including let-7f-5p and hsa-miR-200b-3p. Findings for the EC versus healthy endometrium comparison were validated against TCGA-UCEC sequencing data which confirmed concordant dysregulation of key miRNAs across platforms. Our findings reveal that EC is characterized by widespread miRNA deregulation, with a unique global down-regulation signature in POLE-mutated tumors. These results highlight the potential of miRNAs as complementary biomarkers for classification and potential targets in EC. Full article

Pluripotent stem cells (PSCs) exhibit remarkable self-renewal capacity and differentiation potential, necessitating tight regulation of gene expression at both transcriptional and post-transcriptional levels. Among post-transcriptional mechanisms, RNA turnover and degradation together play pivotal roles in maintaining transcriptome homeostasis and controlling RNA stability. RNA degradation plays a pivotal role in determining transcript stability for both messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs), thereby influencing cell identity and fate transitions. The core RNA decay machinery, which includes exonucleases, decapping complexes, RNA helicases, and the RNA exosome, ensures timely and selective decay of transcripts. In addition, RNA modifications such as 5′ capping and N6-methyladenosine (m⁶A) further modulate RNA stability, contributing to the fine-tuning of gene regulatory networks essential for maintaining PSC states. Recent single-cell and multi-omics studies have revealed that RNA degradation exhibits heterogeneous and dynamic kinetics during cell fate transitions, highlighting its role in preserving transcriptome homeostasis. Conversely, disruption of RNA decay pathways has been implicated in developmental defects and disease, underscoring their potential as therapeutic targets. Collectively, RNA degradation emerges as a central regulator of PSC biology, integrating the decay of both mRNAs and ncRNAs to orchestrate pluripotency maintenance, lineage commitment, and disease susceptibility. Full article

Bacillus subtilis DinG/XPD-like paralogues, DinG and YpvA, have been implicated in overcoming replication stress. DinG possesses a DEDD exonuclease and DNA helicase domains, whereas YpvA lacks the DEDD exonuclease domain. We report that DinG·Mg²⁺ (hereafter referred to as DinG) degrades linear single-stranded (lss) DNA with 3′→5′ polarity and binds lssDNA with higher affinity than its exonuclease-deficient mutant DinG D10A E12A. DinG’s ssDNA-dependent ATPase activity neither stimulates nor inhibits DNA degradation. When bound to the 3′-end of forked DNA, DinG destabilises and degrades the substrate; however, in the presence of ATP, DinG dissociates before reaching the duplex junction. DinG degrades the RNA strand within RNA–DNA hybrids but does not cleave lssRNA unless complexed with Mn²⁺. DinG removes genomic R-loops, as RnhC and PcrA do. DinG physically interacts with RecA and PolA and functions in the same pathway as translesion synthesis (TLS) DNA polymerases (DNAPs) to respond to both spontaneous and methyl methanesulphonate (MMS)-induced mutagenesis. DinG-mGold forms spontaneous foci at or near replication forks, which become enriched following MMS or rifampicin treatment. We propose that DinG contributes to mitigating replication stress by degrading R-loop barriers and facilitating TLS, potentially via RecA-linked mechanisms. Full article

Background/Objectives: Schizophrenia (SCZ) is a highly heritable mental disorder with a complex polygenic genetic architecture. The heat shock protein 90 alpha (HSP90α), encoded by the HSP90AA1 gene, is a molecular chaperone that is required for the proper folding and activity of many of the client proteins that are involved in numerous essential cellular pathways. In addition to its general chaperone activity, HSP90α plays a role in other neuronal contexts and was found to have an altered expression in SCZ, which makes HSP90AA1 an attractive gene for association studies. The aim of this study was to determine whether the HSP90AA1 polymorphisms (rs8005905, rs10873531, rs11621560, rs4947 and rs2298877) are involved in the risk of developing SCZ and its clinical picture in a Polish Caucasian population. Methods: A total of 1088 unrelated subjects (409 patients and 679 healthy controls) were included in the study. The SNPs were genotyped using a TaqMan 5′-exonuclease allelic discrimination assay. The results of the Positive and Negative Syndrome Scale (PANSS) were presented in the five-dimensional model. Results: None of the SNPs were associated with a predisposition to developing SCZ in either the single-marker or haplotype analysis including the results of gender-stratified analyses. However, the genotypes of rs11621560, rs4947 and rs2298877 SNPs were associated with the emotional distress (EMO) dimension score. Conclusions: The results of the present study indicate that HSP90AA1 variants may have an impact on the psychopathology of SCZ, although larger studies are needed to clarify these findings. Full article

Background/Objectives: Phosphoglucomutase-1 (PGM1) is an enzyme that plays important roles in glycolysis, glycogen metabolism, and glycosylation. The PGM1 gene harbors two common nonsynonymous single-nucleotide variants (rs1126728 and rs11208257), which result in four functional PGM1 phenotypes. Correlations between PGM1 polymorphisms and several pathological conditions have been suggested. Methods: To identify the rs1126728 and rs11208257 concurrently, a fluorescence melting curve analysis (FMCA) was developed that utilizes two distinct dual-labeled fluorescence probes. Two distinct Taq polymerases, one with and one without 5′-3′exonuclease activity, were compared. This method was then applied to 95 unrelated Japanese subjects. Results: Both Taq polymerases, with and without 5′-3′exonuclease activity, were found to be sufficiently functional. Furthermore, the results of the FMCA using both Taq polymerases were compared with the direct Sanger sequencing results of PCR products from the 95 Japanese subjects, demonstrating 100% concordance. Conclusions: The duplex probe-based FMCA developed in this study is useful for examining the association between rs1126728 or rs11208257 and a range of pathological conditions using a relatively large number of subjects. Full article

Polymerase-coupled nanopore sequencing requires DNA polymerases with strong strand displacement activity and high processivity to sustain continuous signal generation. In this study, we characterized two novel B family DNA polymerases, SRHS and BBum, isolated from Bacillus phages SRT01hs and BeachBum, respectively. Both enzymes exhibited robust strand displacement, 3′→5′ exonuclease activity, and maintained processivity under diverse reaction conditions, including across a broad temperature range (10–45 °C) and in the presence of multiple divalent metal cofactors (Mg²⁺, Mn²⁺, Fe²⁺), comparable to the well-characterized Phi29 polymerase. Through biochemical analysis of mutants designed using AlphaFold3-predicted structural models, we identified key residues (G96, M97, D486 in SRHS; S97, M98, A493 in BBum) that modulated exonuclease activity, substrate specificity and metal ion utilization. Engineered variants SRHS_F and BBum_Pro_L efficiently incorporated unnatural nucleotides in the presence of Mg²⁺—a function not observed in Phi29 and other wild-type strand-displacing B family polymerases. These combined biochemical features highlight SRHS and BBum as promising enzymatic scaffolds for nanopore-based long-read sequencing platforms. Full article

Circular RNA (circRNA) has gained significant attention as a potential therapeutic tool due to its remarkable stability and resistance to degradation by exonucleases. However, scalable and efficient methods for purification and delivery remain critical challenges that must be addressed. In this study, we developed and evaluated an optimized affinity chromatography method using Oligo (dT) columns for the purification of circRNA, achieving high yield and purity compared with high-performance liquid chromatography. Additionally, we investigated the in vivo efficacy of circRNA-Oligo (dT) encapsulated in lipid nanoparticles (LNPs) formulated with emerging ionizable lipids, including CP-LC-0867 and CP-LC-0729. Our results showed that LNPs formulated with CP-LC-0867 consistently produced higher protein expression compared to SM-102, with sustained luciferase activity observed over a 14-day period. Furthermore, we assessed the lyophilization potential of LNP-circRNA-Oligo (dT) using CP-LC-0729 to extend the shelf life and eliminate the need for ultra-low-temperature storage. Remarkably, the lyophilized LNPs exhibited no significant differences in protein expression compared to their non-lyophilized counterparts, demonstrating that lyophilization is a viable strategy for extending the storage and transport of circRNA therapies. These findings underscore the potential of optimized new ionizable lipids, improved purification strategies, and lyophilization techniques to enhance the scalability, stability, and practical application of circRNA therapies. Full article

Background: Synthetic DNA aptamers are a class of molecules with potential applications in medicine, serving as molecular sensors or ligands for targeted drug delivery. Systematic evolution of ligands by exponential enrichment (SELEX) is a technology for selecting functional aptamers that was first reported three decades ago and has been actively developed since. SELEX involves multiple iterations of two fundamental steps: (i) target affinity-based partitioning of aptamers from a random library and (ii) amplification of selected aptamers by PCR, followed by isolation of single-stranded DNA (ssDNA). SELEX protocols have diversified considerably, with numerous variations possible for each step. This heterogeneity makes it challenging to identify optimal methods. Comparative analysis of different approaches for the major stages of SELEX is therefore of considerable practical importance. Methods: Four widely used methods for ssDNA generation were performed in parallel: (a) PCR followed by digestion of the antisense strand with exonuclease lambda, (b) PCR with an extended primer followed by size-dependent strand separation using denaturing PAGE, (c) asymmetric PCR, and (d) asymmetric PCR with a primer-blocker. Results: The specificity, efficiency, reproducibility, and duration of each method were compared. Conclusions: Asymmetric PCR with a primer-blocker yielded the most favorable results. Full article

Pharmacogenomics is revolutionizing precision medicine by enabling tailored therapeutic strategies based on an individual genetic and molecular profile. Circular RNAs (circRNAs), a distinct subclass of endogenous non-coding RNAs, have recently emerged as key regulators of drug resistance, tumor progression, and therapeutic responses. Their covalently closed circular structure provides exceptional stability and resistance to exonuclease degradation, positioning them as reliable biomarkers and novel therapeutic targets in cancer management. This review provides a comprehensive analysis of the interplay between circRNAs and pharmacogenomics, focusing on their role in modulating drug metabolism, therapeutic efficacy, and toxicity profiles. We examine how circRNA-mediated regulatory networks influence chemotherapy resistance, alter targeted therapy responses, and impact immunotherapy outcomes. Additionally, we discuss emerging experimental tools and bioinformatics techniques for studying circRNAs, including multi-omics integration, machine learning-driven biomarker discovery, and high-throughput sequencing technologies. Beyond their diagnostic potential, circRNAs are being actively explored as therapeutic agents and drug delivery vehicles. Recent advancements in circRNA-based vaccines, engineered CAR-T cells, and synthetic circRNA therapeutics highlight their transformative potential in oncology. Furthermore, we address the challenges of standardization, reproducibility, and clinical translation, emphasizing the need for rigorous biomarker validation and regulatory frameworks to facilitate their integration into clinical practice. By incorporating circRNA profiling into pharmacogenomic strategies, this review underscores a paradigm shift toward highly personalized cancer therapies. circRNAs hold immense potential to overcome drug resistance, enhance treatment efficacy, and optimize patient outcomes, marking a significant advancement in precision oncology. Full article

In this study, novel fluorescent DNA biosensors for mercury (Hg²⁺) and silver (Ag⁺) ions were developed based on thymine (T)- and cytosine (C)-rich recognition elements in combination with exonuclease III and a mismatch-catalyzed hairpin assembly (MCHA)-based cascade isothermal signal-amplification strategy. In the presence of the respective target analytes, the recognition element terminals form so-called T-Hg²⁺-T or C-Ag⁺-C structures, resulting in cleavage by Exo III and the release of the trigger strand for MCHA. This binds to the H1 hairpin, which is fluorescently labeled with carboxyfluorescein (FAM) and tetramethylrhodamine (TAMRA), disrupting fluorescence resonance energy transfer between them and, thus, restoring FAM fluorescence, generating a strong signal at 520 nm. The linear range of the Hg²⁺ sensor is 0.5 to 3 pM, with a detection limit of 0.07 pM. The recovery range in actual spiked water samples is between 98.5% and 105.2%, with a relative standard deviation (RSD) ranging from 2.0% to 4.2%. The linear range of the Ag⁺ sensor is 10 to 90 pM, with a detection limit of 7.6 pM. The recovery range in actual spiked water samples is between 96.2% and 104.1%, with an RSD ranging from 3.2% to 6.3%. The cascade isothermal signal amplification strategy effectively enhances sensor sensitivity, while MCHA decreases the false-positive rate. The aptamer sensor exhibits high specificity, is resistant to interference, and can be used for the detection of Hg²⁺ and Ag⁺ in environmental water samples. Full article

The Bunyavirales order includes a range of zoonotic viruses, which can cause severe disease in humans. The viral replication machinery is a logical target for the development of direct-acting antivirals. Inhibition of the cap-snatching endonuclease activity of related influenza viruses provides a proof of concept. Using the influenza B virus (IBV) RNA-dependent RNA polymerase complex as a benchmark, we conducted a comparative analysis of endonuclease activities of recombinant full-length bunyaviral L proteins using gel-based assays. The IBV complex demonstrates specific endonucleolytic cleavage and a clear preference for capped substrates. In contrast, severe fever with thrombocytopenia syndrome, Sin Nombre, and Hantaan virus L proteins readily cleave capped and uncapped RNAs to a broader spectrum of RNA fragments. Active site mutants further help to control for the potential of contaminating nucleases, exonuclease activity, and RNA hydrolysis. The influenza cap-snatching inhibitor baloxavir and derivatives have been used to validate this approach. In conclusion, the results of this study demonstrate the importance of assays with single nucleotide resolution and the use of full-length L proteins as a valuable experimental tool to identify selective endonuclease inhibitors. Full article