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.

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.

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.

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).

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.

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.

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.

Small Circular DNA Molecules as Triangular Scaffolds for the Growth of 3D Single Crystals.

DNA is a very useful molecule for the programmed self-assembly of 3D (three dimension) nanoscale structures. The organised 3D DNA assemblies and crystals enable scientists to conduct studies for many applications such as enzymatic catalysis, biological immune analysis and photoactivity. The first self-assembled 3D DNA single crystal was reported by Seeman and his colleagues, based on a rigid triangle tile with the tile side length of two turns. Till today, successful designs of 3D single crystals by means of programmed self-assembly are countable, and still remain as the most challenging task in DNA nanotechnology, due to the highly constrained conditions for rigid tiles and precise packing. We reported here the use of small circular DNA molecules instead of linear ones as the core triangle scaffold to grow 3D single crystals. Several crystallisation parameters were screened, DNA concentration, incubation time, water-vapour exchange speed, and pH of the sampling buffer. Several kinds of DNA single crystals with different morphologies were achieved in macroscale. The crystals can provide internal porosities for hosting guest molecules of Cy3 and Cy5 labelled triplex-forming oligonucleotides (TFOs). Success of small circular DNA molecules in self-assembling 3D single crystals encourages their use in DNA nanotechnology regarding the advantage of rigidity, stability, and flexibility of circular tiles.

Synthesis of Ligand Functionalized ErbB-3 Targeted Novel DNA Nano-Threads Loaded with the Low Dose of Doxorubicin for Efficient In Vitro Evaluation of the Resistant Anti-Cancer Activity.

PURPOSE: Doxorubicin (Dox) being a hydrophobic drug needs a unique carrier for the effective encapsulation with uniformity in the aqueous dispersion, cell culture media and the biological-fluids that may efficiently target its release at the tumor site. METHODS: Circular DNA-nanotechnology was employed to synthesize DNA Nano-threads (DNA-NTs) by polymerization of triangular DNA-tiles. It involved circularizing a linear single-stranded scaffold strand to make sturdier and rigid triangles. DNA-NTs were characterized by the AFM and Native-PAGE tests. Dox binding and loading to the Neuregulin1 (NRG1) functionalized DNA based nano-threads (NF-DBNs) was estimated by the UV-shift analysis. The biocompatibility of the blank NRG-1/DNA-NTs and enhanced cytotoxicity of the NF-DBNs was assessed by the MTT assay. Cell proliferation/apoptosis was analyzed through the Flow-cytometry experiment. Cell-surface binding and the cell-internalization of the NF-DBNs was captured by the double-photon confocal microscopy (DPCM). RESULTS: The AFM images revealed uniform DNA-NTs with the diameter 30 to 80 nm and length 400 to 800 nm. PAGE native gel was used for the further confirmation of the successful assembly of the strands to synthesize DNA-NTs that gave one sharp band with the decreased electrophoretic mobility down the gel. MTT assay showed that blank DNA-NTs were biocompatible to the cells with less cytotoxicity even at elevated concentrations with most of the cells (94%) remaining alive compared to the dose-dependent enhanced cytotoxicity of NF-DBNs further evidenced by the Flow-cytometry analysis. CONCLUSION: Uniform and stiffer DNA-NTs for the potential applications in targeted drug delivery was achieved through circular DNA scaffolding.

In Vitro Identification of the Hamiltonian Cycle Using a Circular Structure Assisted DNA Computer.

Adleman's illustration of molecular computing using DNA paved the way toward an entirely new direction of computing (Adleman, L. M. Science 1994, 266, 1021). The exponential time complex combinatorial problem on a traditional computer turns out to be a separation problem involving a polynomial number of steps in DNA computing experiments. Despite being a promising concept, the implementations of existing DNA computing procedures were restricted only to the smaller size formulations. In this work, we demonstrate a structure assisted DNA computing procedure on a bigger size Hamiltonian cycle problem involving 18 vertices. The developed model involves the formation and digestion of circular structure DNA, iteratively over multiple stages to eliminate the incorrect solutions to the given combinatorial problem. A high accuracy is obtained compared to other structure assisted models, which enable one to solve the bigger size problems.

Circular Nucleic Acids: Discovery, Functions and Applications.

Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology-controlled or enabled molecular devices. This article will begin by summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts to couple functional nucleic acids with rolling circle amplification for ultra-sensitive biosensing and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery will be recapitulated.

Circle-Seq: Isolation and Sequencing of Chromosome-Derived Circular DNA Elements in Cells.

Chromosome-derived extrachromosomal circular DNA elements (eccDNAs) are detected in all eukaryotes examined so far. Here I describe the Circle-Seq protocol, applicable for physical enrichment of eccDNAs of a broad size range, combined with sequence confirmation of circular structures.Briefly, by concise alkaline treatment and gentle gravity flow-through an ion-exchange column, eccDNAs are enriched in the eluate fraction. EccDNAs are enzymatically isolated by extensive Plasmid-Safe DNase digestion of linear chromosomes and further enriched by φ29 rolling circle amplification. By means of high throughput sequencing of amplified eccDNA and custom eccDNA mapping software, around ten-thousand unique eccDNA types could be detected at nucleotide resolution in a million human muscle nuclei by this method.