DNA Template Research Articles

Widespread herpesvirus infections are associated with various diseases. DNA replication of human herpes simplex virus type 1 (HSV-1) requires a helicase-primase (HP) complex of three core proteins: UL5, UL52, and UL8. This complex unwinds viral DNA and synthesizes primers for DNA replication, making it an attractive antiviral target. Although HP inhibitors pritelivir and amenamevir were identified through screening, their binding mechanisms remain unclear. Here, we report cryo-electron microscopy structures of HSV-1 HP bound to a forked DNA template alone and in complex with pritelivir or amenamevir. The structures reveal a bilobed architecture highlighting HP coordinated action at the replication fork and providing a structural basis for HP inhibition by illustrating precisely how pritelivir and amenamevir block helicase activity. Data lay a solid foundation for the development of improved antiviral therapies.

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.

PCR-generated DNA templates enable efficient, rapid, and cost-effective mRNA synthesis.

Specific regulation of gene expression via modulation of G-quadruplex stability using reduction-sensitive azobenzene ligands.

CRISPR-Cas9 HDR optimization: RAD52, denatured, and 5′-modified DNA templates in knock-in mice generation

A multiplexed TSA/CRISPR-mediated one-pot system for rapid detection of high-risk animal-derived infectious diseases.

We report a novel cell-free technology, ICED (Intra-Circular Expression and Display), for displaying an expressed protein on its encoding circular DNA. The recovered circular DNA, enriched by affinity-based screening of nascent protein, can be directly amplified using RCR, the reconstituted E. coli replication-cycle reaction. Unlike CIS display, which requires the replication initiator RepA and cis-acting elements including the oriR, the cognate binding site of RepA from the R1 plasmid, ICED does not depend on such a specific protein or DNA elements. The display is abolished by linearization of template DNA, inserting a transcription terminator, or treatment with RNaseA as well as puromycin. Its efficiency is enhanced by the addition of magnesium in the selection step. These suggest that the expressed protein remains anchored to the circular DNA via a transcription-translation (TX-TL) complex involving RNA polymerase, mRNA, and ribosome. This previously unrecognized linkage offers new insight into the mechanistic interface in TX-TL. Notably, the system is compatible not only with crude extracts but also with the reconstituted PURE system composed of E. coli or T7 RNA polymerase and purified translation factors. By directly reusing the selected RCR product for subsequent rounds, we achieved 108-fold enrichment by two rounds, surpassing the performance of conventional display platforms. ICED thus provides a more efficient and straightforward platform for cell-free display.

Detecting protein biomarkers with high sensitivity is essential for early disease diagnosis, treatment optimization, and basic biomedical research. Conventional enzyme-linked immunosorbent assays (ELISA) often lack the sensitivity required for low-abundance protein detection, whereas digital ELISA offers ultrahigh sensitivity but faces limitations in multiplexing and accessibility due to reliance on specialized instrumentation. To address these challenges, we developed FluoMag-dPEA (Fluorescence-coded, Magnetic bead-enhanced digital Proximity Extension Assay), a streamlined platform that integrates magnetic bead-enhanced proximity extension assay with digital PCR (dPCR) for highly sensitive and multiplexed protein detection. FluoMag-dPEA platform employs a ratiometric fluorescence coding scheme to achieve scalable multiplexing that can be decoded with any standard two-color dPCR readouts. Target proteins are converted into fluorescence-coded DNA templates on magnetic beads, which are then released for bead-free digital analysis, ensuring broad compatibility with common dPCR systems. The approach achieves attomolar sensitivity and enables precise, simultaneous quantification of multiple proteins. We validated FluoMag-dPEA using an eight-plex cytokine assay to profile secretions from peripheral blood mononuclear cells, demonstrating strong concordance with the benchmark Luminex multiplex protein assay in measuring secreted cytokines while achieving superior sensitivity at lower concentrations, reliably detecting proteins at single-cell equivalents. By offering ultrahigh sensitivity, scalable multiplexing, and accessibility, FluoMag-dPEA represents a powerful tool for protein biomarker detection and holds significant promise for diverse biomedical and clinical applications by many users.

Although progress has been seen for low template DNA (LT-DNA) analysis in the last few years, the obtainment of full short tandem repeat (STR) profiles remains challenging in forensic genetics. Preamplification treatment could help increase the recovery of genetic information in STR typing. In this study, an efficient amplification method was investigated, which shows that the application of the abasic site in a single primer for targets could efficiently delete the primer-binding site of PCR products to achieve semi-linear amplification (abSLA PCR). Further, the position of abasic sites located at 8th to 10th from the 3’ end of the primers could facilitate PCR amplification. A significant increase in the recovery of STR loci could be obtained using a 4-plex STR pre-amplification (D8S1179, D21S11, D7S820, and CSF1PO) coupled to Identifiler Plus kit in low template of genomic DNA or single cells. Altogether, the application of abSLA amplification method can facilitate conventional STR typing in LT-DNA analysis.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19847-1.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are versatile emitters whose photophysical properties are defined by the DNA template. In this study, we explore the unusual temperature- and concentration-dependent behavior of two NIR-emissive DNA-AgNCs: one stabilized by two 16-base DNA oligomers (16mer), and another embedded within a 3-base shortened version of these strands (13mer). Using a combination of optical spectroscopy and mass spectrometry, we show that Ag+-carrying DNA strands can reversibly attach to DNA-AgNCs and fine-tune their photophysical properties. For 13mer-AgNC, we demonstrate the presence of three different species, (13mer)2-[Ag20]10+, (13mer)3-[Ag27]17+, and (13mer)4-[Ag34]24+, depending on concentration and temperature. For the AgNC stabilized by two 16-base DNA strands, (16mer)2-[Ag20]10+, emission wavelength and intensity vary with temperature, although no significant concentration-dependent spectral shifts are observed. While mass-spectrometry revealed the existence of (16mer)3-[Ag27]17+, time-resolved anisotropy uncovered the formation of aggregates of (16mer)2-[Ag20]10+. These findings demonstrate the presence of dynamic equilibria between Ag+-carrying DNA strands and DNA-AgNCs.

Islatravir (ISL, EFdA) is a nucleoside analog that inhibits HIV-1 reverse transcriptase (RT) translocation during viral replication. Its high potency stems from unique structural features: a 4'-ethynyl group that interacts with the hydrophobic pocket (containing A114, Y115, F160, M184, and D185) in HIV-1 RT, hindering translocation, and a 3'-hydroxyl group that mimics natural nucleosides for efficient incorporation. Recent phase 3 clinical trials, combining ISL with Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, show that it is noninferior to existing treatments, offering a unique advantage due to their distinct resistance profiles. For instance, DOR-associated mutations, V106I/F227C, which confer >105-fold DOR resistance in clinics, unexpectedly boost ISL's potency by 2.3-fold in published cell-based resistance selection assays. In contrast, V106I alone does not affect Islatravir's potency, while F227C alone enhances it by 5.6-fold. To kinetically understand these findings, we used presteady-state kinetic assays to determine the kinetic parameters for EFdA 5'-triphosphate (EFdA-TP) and dATP incorporation. We found that the incorporation efficiency of EFdA-TP was 1.4-fold higher than that of dATP on an RNA template and 1.7-fold higher on a DNA template with the F227C mutant. However, this difference was only 1.1- to 1.3-fold higher with the F227C/V106I mutant. Our energy-minimized modeling revealed that these mutations remotely alter the hydrophobic 4'-ethynyl group-binding pocket's structure, surprisingly strengthening the pocket's binding interactions with EFdA-TP. Alongside this, the F227C mutation decreased dATP's binding affinity with both templates. Our data established a kinetic basis for the published cell-based resistance selection assay results, underscoring the significant potential of the ISL/DOR combination therapy in treating HIV-1 infected patients.

Abstract Amplifying long and complex genomic regions with high fidelity is essential for accurate long-read sequencing, especially in diseases linked to repeat expansions, where polymerase slippage and stalling can compromise sequencing results, ultimately impacting disease diagnosis and prognosis. In Fragile X Syndrome, for example, amplifying the FMR1 gene is challenging due to CGG repeat expansions that often exceed 200 repeats. Also, Short-read Next Generation Sequencing struggles to detect these expansions accurately, relying on computational algorithms for sequence assembly, this can reduce sequence and, therefore, diagnostic accuracy. Consequently, long-read sequencing offers a more reliable method for directly identifying longer sequences, with the amplification step being crucial to ensuring successful detection. We evaluated the performance of DeCodi-Fi, a novel high-fidelity polymerase, by amplifying and sequencing genes with tandem repeats linked with the following disease-associated genes: FMR1 (Fragile X Syndrome), C9orf72 (Amyotrophic Lateral Sclerosis, ALS), IT15 (Huntington*s Disease), and X25 (Friedreich*s Ataxia), as well as long templates exceeding 12 kb, including BRCA1 (Breast Cancer) and SMN1 (Spinal Muscular Atrophy) in previously reported healthy and diagnosed individuals. Sequencing analysis was performed to assess DeCodi-Fi*s accuracy and ability to amplify complex templates without introducing errors. Its performance was also compared against other commercial high-fidelity polymerase. We successfully optimized PCR conditions for four repeat expansion targets and two long amplicons of clinical relevance. Long-read sequencing libraries were generated and analyzed using Oxford Nanopore technology. Consensus sequences were mapped to curated data from the corresponding Coriell samples. DeCodi-Fi polymerase successfully amplified repeat expansions with +/- 1 repeat accuracy. For the non-repeated long amplicons we obtained full coverage and polymerase error rates below the sequencing error threshold, when compared to the repository sequence data. This study demonstrates that DeCodi-Fi is capable of amplifying complex and long DNA templates with minimal errors, providing a reliable tool for long-read sequencing involving challenging genomic regions.

Deoxyribozymes that ligate DNA will expand the reaction scope of DNA catalysis and are useful in the construction of DNA nanostructures. Herein, we report the first efforts to isolate a novel class of DNA ligase deoxyribozymes from a random sequence DNA pool by in vitro selection. The identified deoxyribozymes catalyze the intermolecular linear DNA-DNA ligation via the formation of unnatural triazole linkages between a 5′ alkyne and a 3′ azide. One remarkable click-ligating deoxyribozyme, named CLDz2, ligated DNA with an observed rate constant (kobs) up to 2.7 × 10−2 h−1 at 10 mM Mn2+ (pH 7.0, 30°C), with up to 40% yield in overnight incubations. CLDz2 is predicated to have a four-way, junction-like structure comprised of four short duplexes, three hairpin loops, and two main interhelical unpaired elements. Comprehensive nucleotide covariation experiments suggest that CLDz2 should be generally applicable for click ligation of a wide range of 3′ azide DNAs. We further demonstrate a CLDz2-directed chemical ligation strategy for the synthesis of single-stranded monomeric circular DNA in high selectivity (97%), which can be used as a DNA template in rolling circle amplification.

Biochemical reconstitution of sister chromatid cohesion establishment during DNA replication.

Cell-free protein synthesis is an in vitro transcription and translation process that takes advantage of the fact that cell growth is dissociated from protein production. After cell cultures have grown to the optimal cell density, the cells are lysed while preserving the cellular machinery so that they can be used in an in vitro protein synthesis reaction. The system uses three main components: cell lysate, a reaction mix, and plasmid DNA. Protein production can be performed in hours on a lab bench, expanding the possibility to evaluate multiple plasmids at once without the need to transform and select positively expressing clones.The use of Komagataella phaffii to generate the lysate for cell-free protein synthesis has been shown with a number of different strains, including a ribosome-overexpressing strain, FHL1. The high cell densities result in a highly active lysate that has been shown to be capable of producing a variety of products, including human serum albumin and virus-like particles (VLPs). Furthermore, as a eukaryotic platform, the system is capable of performing post-transcriptional modifications such as creating disulfide bonds without the need for additional folding chaperones or supplements. Here, we describe the methods required for generating cell lysate, creating a reaction mix that has been optimized through design of experiments, optimizing the concentration of DNA template, and using a reporter protein to monitor expression yields. Harnessing the potential of CFPS using K. phaffii can lead to faster prototyping of vectors before strain construction, facilitating the rapid identification of optimal protein variants. It can also be used to produce proteins that are toxic to living cells, providing an alternative source of proteins for experimentation.

Comprehensive review and meta-analysis of magnesium chloride optimization in PCR: Investigating concentration effects on reaction efficiency and template specificity.

Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis.

Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an exvivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective invivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.

Hepatitis B virus (HBV) is a small DNA virus that establishes chronic infection and drives progressive liver disease and cancer; presenting a global health problem with more than 250 million infections. HBV replicates via an episomal covalently-closed-circular DNA (cccDNA) and integrated viral DNA fragments are linked to carcinogenesis. Current treatments only suppress HBV replication and there is a global initiative to develop genome targeting therapies, including siRNAs, antisense oligonucleotides and epigenetic modifiers specific for HBV cccDNA. However, our knowledge of the cccDNA and integrant transcriptomes is confounded by overlapping viral RNAs. Using targeted long-read sequencing we mapped the HBV transcriptome in liver biopsies from eleven treatment naïve patients. Probe enrichment yielded robust sequencing libraries and identified cccDNA-derived genomic and sub-genomic transcripts, and a repertoire of previously uncharacterised spliced, truncated and chimeric viral RNAs. Assigning viral transcripts to their respective DNA templates revealed differential promoter activity in cccDNA and integrants, with implications for the efficacy of epigenetic modifiers. Integrant-derived transcripts showed vast diversity in the viral-host junctions, posing a challenge for current nucleotide-targeting therapies. cccDNA was a source of genetic polymorphism, with distinct viral lineages present in the surface antigen encoding region, providing an insight into hepadnavirus evolution during chronic infection.

Mutations that impact subunits of mammalian SWI/SNF (mSWI/SNF or BAF) chromatin remodeling complexes are found in over 20% of human cancers. Among these subunits, AT-rich interactive domain-containing protein 1A (ARID1A) is the most frequently mutated gene, occurring in over 8% of various cancers. The majority of ARID1A mutations are frameshift or nonsense mutations, causing loss of function. Previous studies have suggested that ARID1A may facilitate interactions between BAF complexes and various transcriptional coactivators, but a biochemical role for ARID1A in BAF remodeling activity has not been identified. Here, we describe the in vitro reconstitution of the cBAF, PBAF, and ncBAF complexes, and we compare their biochemical activities. In addition, we reconstitute a variety of cBAF subcomplexes, defining roles for several subunits in high affinity nucleosome binding and nucleosome sliding activity. Remarkably, we find that the ARID1A subunit of cBAF is largely dispensable for nucleosome binding, nucleosome sliding, and adenosine triphosphatase activity, but ARID1A is required for cBAF to transfer histone octamers between DNA templates. Our study reveals a biochemical function of ARID1A/ARID1B in BAF-mediated chromatin remodeling, suggesting a model in which dysregulation of histone octamer transfer activity of BAF complexes may be relevant to cancer formation.