Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick.

The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2-4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5-bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are also helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.

A standardized protocol for sample preparation for scanning electron microscopy to visualize extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) are circular DNA structures associated with cancer and drug resistance. One specific type, double minute (DM) chromosomes, has been studied since the 1960s using imaging techniques like cytogenetics and fluorescence microscopy. Specialized techniques such as atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer micro to nano-scale visualization, but current sample preparation methods may not optimally preserve ecDNA structure. Our study introduces a systematic protocol using SEM for high-resolution ecDNA visualization. We have optimized the end-to-end procedure, providing a standardized approach to explore the circular architecture of ecDNA and address the urgent need for better understanding in cancer research.

Despite advances in extrachromosomal DNA (ecDNA) detection, current methods struggle to reveal ecDNA's architecture within cells. Specialized techniques like scanning electron microscopy (SEM) provide the needed resolution, but existing sample preparation may not preserve ecDNA well. Our study introduces a systematic method using SEM, optimizing procedures for preparing and visualizing metaphase spread samples. This offers a standardized approach to study ecDNA's circular architecture, addressing a pressing need in cancer research.

Progress in application of cyclic single-stranded nucleic acids.

Cyclic nucleic acids are biologically stable against nucleic acid exonucleases due to the absence of 5' and 3' termini. Studies of cyclic nucleic acids mainly focus on cyclic single-stranded nucleic acids. Cyclic single-stranded nucleic acids are further divided into circular RNA (circRNA) and circular single-stranded DNA (cssDNA). The synthesis methods of circRNA include lasso-driven cyclization, intron-paired cyclization, intron cyclization, intron complementary pairing-driven cyclization, RNA-binding protein-driven cyclization, and artificial synthesis depending on the source. Its main role is to participate in gene expression and the treatment of some diseases. Circular single-stranded DNA is mainly synthesized by chemical ligation, template-directed enzyme ligation, and new techniques for the efficient preparation of DNA single loops and topologies based on CircLigase. It is mainly used in rolling circle amplification (RCA) technology and in the bioprotection of circular aptamers and second messengers. This review focuses on the types, synthesis methods, and applications of cyclic single-stranded nucleic acids, providing a reference for further research on cyclic single-stranded nucleic acids.

Large bubble drives circular DNA melting.

We investigate the melting transition of non-supercoiled circular DNA of different lengths, employing Brownian dynamics simulations. In the absence of supercoiling, we find that melting of circular DNA is driven by a large bubble, which agrees with the previous predictions of circular DNA melting in the presence of supercoiling. By analyzing sector-wise changes in average base-pair distance, our study reveals that the melting behavior of circular DNA closely resembles that of linear DNA. Additionally, we find a marked difference in the thermal stability of circular DNA over linear DNA at very short length scales, an effect that diminishes as the length of circular DNA increases. The stability of smaller circular DNA is linked to the occurrence of transient small bubbles, characterized by a lower probability of growth.

From Bioreactor to Bulk Rheology: Achieving Scalable Production of Highly Concentrated Circular DNA.

DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high-throughput screening. To address this, recent advancements in bioreactor-based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram-scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116 mg mL-1. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time-concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.

Microfluidic isolation of extrachromosomal circular DNA through selective digestion of plasmids and linear DNA using immobilized nucleases.

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.

MSC microvesicles loaded G-quadruplex-enhanced circular single-stranded DNA-9 inhibits tumor growth by targeting MDSCs.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) promote tumor growth, metastasis, and lead to immunotherapy resistance. Studies revealed that miRNAs are also expressed in MDSCs and promote the immunosuppressive function of MDSCs. Currently, few studies have been reported on inducible cellular microvesicle delivery of nucleic acid drugs targeting miRNA in MDSCs for the treatment of malignant tumors. RESULTS AND CONCLUSION: In this study, we designed an artificial DNA named G-quadruplex-enhanced circular single-stranded DNA-9 (G4-CSSD9), that specifically adsorbs the miR-9 sequence. Its advanced DNA folding structure, rich in tandem repeat guanine (G-quadruplex), also provides good stability. Mesenchymal stem cells (MSCs) were prepared into nanostructured vesicles by membrane extrusion. The MSC microvesicles-encapsulated G4-CSSD9 (MVs@G4-CSSD9) was delivered into MDSCs, which affected the downstream transcription and translation process, and reduced the immunosuppressive function of MDSCs, so as to achieve the purpose of treating melanoma. In particular, it provides an idea for the malignant tumor treatment.

Surface-Based Multimeric Aptamer Generation and Bio-Functionalization for Electrochemical Biosensing Applications.

Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.

DNA sequence and methylation prescribe the inside-out conformational dynamics and bending energetics of DNA minicircles.

Eukaryotic genome and methylome encode DNA fragments' propensity to form nucleosome particles. Although the mechanical properties of DNA possibly orchestrate such encoding, the definite link between 'omics' and DNA energetics has remained elusive. Here, we bridge the divide by examining the sequence-dependent energetics of highly bent DNA. Molecular dynamics simulations of 42 intact DNA minicircles reveal that each DNA minicircle undergoes inside-out conformational transitions with the most likely configuration uniquely prescribed by the nucleotide sequence and methylation of DNA. The minicircles' local geometry consists of straight segments connected by sharp bends compressing the DNA's inward-facing major groove. Such an uneven distribution of the bending stress favors minimum free energy configurations that avoid stiff base pair sequences at inward-facing major grooves. Analysis of the minicircles' inside-out free energy landscapes yields a discrete worm-like chain model of bent DNA energetics that accurately account for its nucleotide sequence and methylation. Experimentally measuring the dependence of the DNA looping time on the DNA sequence validates the model. When applied to a nucleosome-like DNA configuration, the model quantitatively reproduces yeast and human genomes' nucleosome occupancy. Further analyses of the genome-wide chromatin structure data suggest that DNA bending energetics is a fundamental determinant of genome architecture.

Rolling circle amplification (RCA)-based DNA hydrogel.

DNA hydrogels have unique properties, including sequence programmability, precise molecular recognition, stimuli-responsiveness, biocompatibility and biodegradability, that have enabled their use in diverse applications ranging from material science to biomedicine. Here, we describe a rolling circle amplification (RCA)-based synthesis of 3D DNA hydrogels with rationally programmed sequences and tunable physical, chemical and biological properties. RCA is a simple and highly efficient isothermal enzymatic amplification strategy to synthesize ultralong single-stranded DNA that benefits from mild reaction conditions, and stability and efficiency in complex biological environments. Other available methods for synthesis of DNA hydrogels include hybridization chain reactions, which need a large amount of hairpin strands to produce DNA chains, and PCR, which requires temperature cycling. In contrast, the RCA process is conducted at a constant temperature and requires a small amount of circular DNA template. In this protocol, the polymerase phi29 catalyzes the elongation and displacement of DNA chains to amplify DNA, which subsequently forms a 3D hydrogel network via various cross-linking strategies, including entanglement of DNA chains, multi-primed chain amplification, hybridization between DNA chains, and hybridization with functional moieties. We also describe how to use the protocol for isolation of bone marrow mesenchymal stem cells and cell delivery. The whole protocol takes ~2 d to complete, including hydrogel synthesis and applications in cell isolation and cell delivery.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Insight into formation propensity of pseudocircular DNA G-hairpins.

We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Gel Electrophoresis Analysis of rDNA Instability in Saccharomyces cerevisiae.

The ribosomal RNA (rDNA) sequence is the most abundant repetitive element in the budding yeast genome and forms a tandem cluster of ~100-200 copies. Cells frequently change their rDNA copy number, making rDNA the most unstable region in the budding yeast genome. The rDNA region experiences programmed replication fork arrest and subsequent formation of DNA double-strand breaks (DSBs), which are the main drivers of rDNA instability. The rDNA region offers a unique system to understand the mechanisms that respond to replication fork arrest as well as the mechanisms that regulate repeat instability. This chapter describes three methods to assess rDNA instability.

Chromothripsis drives the evolution of gene amplification in cancer.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.

Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection.

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.

New Insights on Molecular Mechanism of Hepatitis B Virus Covalently Closed Circular DNA Formation.

The chronic factor of the Hepatitis B Virus (HBV), specifically the covalently closed circular DNA (cccDNA), is a highly stable and active viral episomal genome established in the livers of chronic hepatitis B patients as a constant source of disease. Being able to target and eliminate cccDNA is the end goal for a genuine cure for HBV. Yet how HBV cccDNA is formed from the viral genomic relaxed circular DNA (rcDNA) and by what host factors had been long-standing research questions. It is generally acknowledged that HBV hijacks cellular functions to turn the open circular DNA conformation of rcDNA into cccDNA through DNA repair mechanisms. With great efforts from the HBV research community, there have been several recent leaps in our understanding of cccDNA formation. It is our goal in this review to analyze the recent reports showing evidence of cellular factor's involvement in the molecular pathway of cccDNA biosynthesis.

Evolution of a highly functional circular DNA aptamer in serum.

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem-loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.

Remarkable Enhancement of Nucleotide Excision Repair of a Bulky Guanine Lesion in a Covalently Closed Circular DNA Plasmid Relative to the Same Linearized Plasmid.

The excision of DNA lesions by human nucleotide excision repair (NER) has been extensively studied in human cell extracts. Employing DNA duplexes with fewer than 200 bp containing a single bulky, benzo[a]pyrene-derived guanine lesion (B[a]P-dG), the NER yields are typically on the order of ∼5-10%, or less. Remarkably, the NER yield is enhanced by a factor of ∼6 when the B[a]P-dG lesion is embedded in a covalently closed circular pUC19NN plasmid (contour length of 2686 bp) rather than in the same plasmid linearized by a restriction enzyme with the B[a]P-dG adduct positioned at the 945th nucleotide counted from the 5'-end of the linearized DNA molecules. Furthermore, the NER yield in the circular pUC19NN plasmid is ∼9 times greater than in a short 147-mer DNA duplex with the B[a]P-dG adduct positioned in the middle. Although the NER factors responsible for these differences were not explicitly identified here, we hypothesize that the initial DNA damage sensor XPC-RAD23B is a likely candidate; it is known to search for DNA lesions by a constrained one-dimensional search mechanism [Cheon, N. Y., et al. (2019) Nucleic Acids Res. 47, 8337-8347], and our results are consistent with the notion that it dissociates more readily from the blunt ends than from the inner regions of linear DNA duplexes, thus accounting for the remarkable enhancement in NER yields associated with the single B[a]P-dG adduct embedded in covalently closed circular plasmids.

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.

Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.