Minicircle DNA Research Articles

SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.

Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT>3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7%) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.

Binding of Histone H1 to DNA Is Differentially Modulated by Redox State of HMGB1

HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

Detection and Characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR Green-Based Real-Time PCR and High Resolution Melt Analysis Targeting Kinetoplast Minicircle DNA

Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples, must be taken into account in quantitative PCR-based applications; however, it might also be used to differentiate between Leishmania subgenera.

The role of sigmodontine rodents as sylvatic hosts of Trypanosoma cruzi in the Argentinean Chaco

The role of rodents in the sylvatic transmission of Trypanosoma cruzi has seldom been investigated using parasitological and molecular methods. We assessed the occurrence of T. cruzi in wild small rodents from Pampa del Indio, in the Argentinean Chaco, and identified the taxonomic status of positive rodents by sequencing a fragment of cytochrome b gene (cytb) and performing BLAST searches and phylogenetic analyses. A total of 176 Sigmodontinae rodents was captured in six surveys using 5425 trap-nights in a wide range of sylvatic habitats between 2009 and 2011. Host infection was determined by xenodiagnosis and by polymerase chain reaction amplification of the hyper-variable region of kinetoplast DNA minicircles of T. cruzi (kDNA-PCR) from blood samples. None of the 176 rodents examined was xenodiagnosis-positive. The prevalence of infection determined by kDNA-PCR from blood samples was 16.2% (95% confidence interval, 10.1–21.9%). Half of the infections detected by kDNA-PCR were confirmed by nuclear satellite DNA-PCR or by kDNA-PCR of the rectal contents of xenodiagnostic bugs. The 24 positive specimens were assigned to eight species, providing the first records of T. cruzi in Akodon montensis, Akodon toba, Graomys chacoensis, and Oligoryzomys chacoensis. The occurrence of T. cruzi infection in Oligoryzomys nigripes, Calomys callosus, Necromys lasiurus and Oecomys sp. (most probably Oecomys mamorae) from the Gran Chaco is also reported for the first time. Although sigmodontine rodents were frequently infected, the intensity of bug rectal infection with T. cruzi was below the detection limit of xenodiagnosis (subpatent infectiousness to bugs), indicating they had a low reservoir host competence.

Understanding How Proteins Shape DNA Using Energy Minimization

Several proteins are known to produce significant changes in DNA shape by locally deforming the double helix or by mediating the formation a loop. For example, the binding of the bacterial proteins HU or Hbb produces large bends in DNA and the tetrameric Lac repressor protein induces the formation of a loop between two distant DNA operators. In order to understand how such proteins sculpt DNA, we have developed a novel optimization method for DNA at the base-pair level. Our method accounts for the sequence-dependent elasticity of DNA and can be applied to a DNA fragment in which the first and last base pairs are spatially constrained. Moreover, our approach makes it possible to constrain intervening parts of the DNA in order to model the presence of bound proteins. We can therefore compute the energy landscape for a wide variety of protein-DNA systems. For example, we studied the effect of the presence of multiple HU or Hbb proteins on DNA minicircles and identified the most likely configurations for given DNA chain lengths and numbers of bound proteins. We also investigated how the presence of HU can enhance or diminish the apparent flexibility of DNA. In addition, we performed a detailed analysis of the energy landscape for loops induced by the Lac repressor. We focused on the binding of the Lac repressor protein on DNA minicircles and studied the competition between large and small loops on supercoiled molecules. We also found that changes in the repressor structure can lead to different loop topologies. Our method makes it possible to conduct thorough analyses of the geometry and mechanics of protein-DNA systems by computing the associated energy landscape and paves the way for the study of other biologically relevant system such as the SV40 minichromosome.

Twisting and bending stress in DNA minicircles.

The interplay between bending of the molecule axis and appearance of disruptions in circular DNA molecules, with ∼100 base pairs, is addressed. Three minicircles with different radii and almost equal contents of AT and GC pairs are investigated. The DNA sequences are modeled by a mesoscopic Hamiltonian which describes the essential interactions in the helix at the level of the base pair and incorporates twisting and bending degrees of freedom. Helix unwinding and bubble formation patterns are consistently computed by a path integral method that sums over a large number of molecule configurations compatible with the model potential. The path ensembles are determined, as a function of temperature, by minimizing the free energy of the system. Fluctuational openings appear along the helix to release the stress due to the bending of the molecule backbone. In agreement with the experimental findings, base pair disruptions are found with larger probability in the smallest minicircle of 66 bps whose bending angle is ∼6°. For this minicircle, a sizeable untwisting is obtained with the helical repeat showing a step-like increase at T = 315 K. The method can be generalized to determine the bubble probability profiles of open ends linear sequences.

Durable expression of minicircle DNA-liposome-delivered androgen receptor cDNA in mice with hepatocellular carcinoma.

Background. The most common gene-based cancer therapies involve the suppression of oncogenic molecules and enhancement of the expression of tumor-suppressor genes. Studies in noncancer disease animal models have shown that minicircle (MC) DNA vectors are easy to deliver and that the proteins from said MC-carrying DNA vectors are expressed over a long period of time. However, delivery of therapeutic genes via a liposome-mediated, MC DNA complex has never been tested in vascular-rich hepatocellular carcinoma (HCC). Liposome-mediated DNA delivery exhibits high in vivo transfection efficiency and minimal systemic immune response, thereby allowing for repetitive interventions. In this study, we evaluated the efficacy of delivering an MC-liposome vector containing a 3.2 kb androgen receptor (AR; HCC metastasis suppressor) cDNA into Hepatitis B Virus- (HBV-) induced HCC mouse livers. Results. Protein expression and promoter luciferase assays revealed that liposome-encapsulated MC-AR resulted in abundant functional expression of AR protein (100 kD) for up to two weeks. The AR cDNA was also successfully delivered into normal livers and diseased livers, where it was persistently expressed. In both normal livers and livers with tumors, the expression of AR was detectable for up to 60 days. Conclusion. Our results show that an MC/liposome delivery system might improve the efficacy of gene therapy in patients with HCC.

Illegal waste sites as potential micro foci of Mediterranean Leishmaniasis

Apart from being against the law, illegal waste dumping also poses a threat to human health and to the environment. Solid and decomposing waste is an ideal breeding ground for a number of rodents, insects, and other vermin that pose a health risk through the spread of infectious diseases. The main objective of this study was to survey disease vectors and rodents for the presence of Leishmania sp. from illegal waste sites along the Istrian Peninsula in Slovenia and Croatia. The entomological and rodent survey was carried out between April 2011 and May 2013, at 12 locations, considering only illegal waste sites which consist of at least 2 m3 of garbage. A total of 119 specimens of Phlebotomine sandflies were collected. Five species were identified as follow: Sergentomyia minuta (48.7%), Phlebotomus perniciosus (30.3%), P. papatasi (13.4%), P. neglectus (5%) and P. mascitii (2.6%). Additionally, 173 small rodents were trapped at the same sites including following species: Rattus rattus (3.5%), Mus musculus (44%), Apodemus agrarius (27%), A. flavicollis (15%) and A. sylvaticus (10.5%). A geospatial analysis software ArcView was used to map the distribution of both vectors and rodents. Sandflies and rodents were screened using a molecular probe to amplify an approximately 120 bp fragment of the kinetoplast DNA (kDNA) minicircle for the detection of Leishmania spp. parasites. While not recorded in the tested sandflies, L. infantum DNA was detected in the spleen of one juvenile black rat (R. rattus). Despite few published records on Leishmania spp. infection in black rats, the addition of our record highlights the importance for further investigation into the frequency and distribution of such occurrences so that we may better classify the role of rodents as potential reservoirs of leishmaniasis in the Mediterranean basin.

Improved Minicircle DNA Biosynthesis for Gene Therapy Applications

Minicircular DNA (mcDNA) biopharmaceuticals have recently risen as a valuable alternative for the development of a next generation of bioactive therapeutics because they are more efficient and safer than standard plasmid DNA (pDNA). To date, the relatively insufficient knowledge regarding mcDNA biosynthesis is currently hindering its manufacture in suitable amounts for clinical trial evaluations. Addressing this limitation is therefore mandatory to bring forth the full therapeutic potential of this cutting-edge technology. Herein, we describe for the first time new processing parameters that improve the overall yield of mcDNA obtained from bacterial fermentations. We provide details for further in-line monitoring and optimization in view of the current good manufacturing guidelines. Our results show that by rising growth temperature to 42°C, an increase in the overall minicircle producer plasmid yield is attained, while biomass amounts are reduced. Moreover, by monitoring in real time the dynamic recombination of parental plasmids to mcDNA, we found that this event is more efficient at specific time points, regardless of the growth temperature and inductor concentration used. These are important findings since mcDNA can be recovered with higher yields at these determined key stages. Indeed, the manipulation of these parameters resulted in a 2.21-fold increase in mcDNA production compared with the established growth temperatures for this technology. Overall, our findings highlight that to achieve maximum productivity while attaining pharmaceutical-grade mcDNA preparations, process design and biosynthesis optimization must take into account key parameters such as temperature, inductor concentration, and recovery time.

In VivoElectroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.

Nonviral delivery for reprogramming to pluripotency and differentiation

Nonviral delivery is a promising strategy for cellular reprogramming to produce desired cell types from undifferentiated stem cells or terminally differentiated somatic cells. Nonviral delivery of genes (DNA, RNA), proteins, or peptides has the potential to reprogram somatic cells to pluripotent stem cells or other lineage cells, and to promote the differentiation of stem cells to specific lineages. Various delivery carriers (cationic polymers, lipids, scaffolds, transposons, cell-penetrating peptides), cargos (episomal plasmids, minicircle DNA, small interfering RNAs, microRNAs, proteins, peptides), and method (electroporation) have been reported. In this article, we review recent advances in nonviral delivery approaches for reprogramming cells to pluripotency or lineage specification.

Subcellular localization of minicircle DNA in the dinoflagellate Amphidinium massartii

SummaryPeridinin‐containing dinoflagellates have small circular DNA molecules called minicircle DNAs, each of which encodes one, or occasionally a few, plastid proteins or ribosomal RNA. Dinoflagellate minicircle DNA is composed of two parts: a gene‐coding sequence and a non‐coding sequence that consists of several variable and core regions. The core regions are identical among the minicircle DNAs with different genes within a species or strain. Because such structure is very different from those of well known plastid DNAs, many functional and evolutionary questions have been raised for the minicircle DNAs, and several studies that focus on answering those questions are underway. However, the localization of minicircle DNA is still controversial: several lines of indirect evidence have implied plastid localization, whereas the nuclear localization of minicircle DNA has also been suggested in a species. In order to understand the evolution and function of minicircle DNA, it is important to know its precise localization. In this study, we sequenced two typical minicircle DNAs, one encodes psbA and the other encodes 23S rRNA genes, from an Amphidinium massartii strain (TM16). To determine the subcellular localization of these minicircle DNAs, we performed DNA‐targeted whole cell fluorescence in situ hybridization with A. massartii minicircle DNA‐specific probes and demonstrated that minicircle DNAs were present in plastids. This study provides the first direct evidence for the plastid localization of dinoflagellate minicircle DNAs.

Detection of Leishmania infantum in 4 different dog samples by real-time PCR and ITS-1 nested PCR

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil, which involves the elimination of infected dogs, the main animal reservoir host of the disease. The aim of the present study was to evaluate a sensitive real-time PCR method for Leishmania infantum detection in 4 different clinical samples of dogs, including the noninvasive conjunctival swab (CS) sample. The results of real-time PCR were compared with those obtained using internal transcribed spacer 1 nested PCR. Animals were divided into 2 groups based on the absence or presence of CVL clinical sings. The CS associated with real-time PCR, using primers addressed to kinetoplast DNA minicircles, was able to detect L. infantum infection in 96.7% of dogs without clinical signs and in 100% of the symptomatic animals, demonstrating the importance of these procedures for diagnosing CVL.

A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles

Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples.AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs–probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The method's minimum detection limit was defined to 11.5ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples.The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority.

Comparison of Standard Plasmid DNA Versus Minicircle DNA for Ultrasound-Mediated Gene Delivery

Comparison of Standard Plasmid DNA Versus Minicircle DNA for Ultrasound-Mediated Gene Delivery

Enhanced Expression of Endothelial Nitric Oxide Synthase in Late Outgrowth Endothelial Progenitor Cells Using Non-Viral Minicircle Dna

Enhanced Expression of Endothelial Nitric Oxide Synthase in Late Outgrowth Endothelial Progenitor Cells Using Non-Viral Minicircle Dna

Percolation phenomena in disordered topological networks

Topological polymer networks consist of circular polymers that are topologically linked. Topological networks made of small circular DNA or protein molecules are of great interest in biology and nanotechnology because they are found in living organisms and can be constructed in-vitro. The physical factors that determine the topology of a network as well as the pathways that are followed for its formation remain poorly understood. In our previous work we proposed a novel biophysical/computational approach to model the formation of planar DNA minicircle networks in trypanosomatid parasites. This model suggests that minicircle networks in trypanosomatid parasites emerged from topologically free minicircles upon space confinement through a percolation pathway. Our model however is somewhat idealized because it assumes that the centers of the minicircles in the network are positioned following a regular planar lattice. Here we propose an extension of the model by allowing the centers of the minicircles to be randomly displaced from the vertices of the lattice. We numerically show that networks form following a percolation pathway upon increasing minicircle density. Our model suggests that the critical percolation density increases as Dperc = 0.8357 − 1.4297 exp(0.6439x) with x is the maximum displacement of the centers of the minicircles. Our results therefore show that the plane distribution of minicircles does not dramatically affect the percolation of minicircles and therefore supports they hypothesis that DNA minicircle networks in trypanosomes evolved through a percolation pathway.

Minicircle DNA is Superior to Plasmid DNA in Eliciting Antigen-specific CD8+ T-cell Responses

Clinical trials reveal that plasmid DNA (pDNA)-based gene delivery must be improved to realize its potential to treat human disease. Current pDNA platforms suffer from brief transgene expression, primarily due to the spread of transcriptionally repressive chromatin initially deposited on plasmid bacterial backbone sequences. Minicircle (MC) DNA lacks plasmid backbone sequences and correspondingly confers higher levels of sustained transgene expression upon delivery, accounting for its success in preclinical gene therapy models. In this study, we show for the first time that MC DNA also functions as a vaccine platform. We used a luciferase reporter transgene to demonstrate that intradermal delivery of MC DNA, relative to pDNA, resulted in significantly higher and persistent levels of luciferase expression in mouse skin. Next, we immunized mice intradermally with DNA encoding a peptide that, when presented by the appropriate major histocompatibility complex class I molecule, was recognized by endogenous CD8(+) T cells. Finally, immunization with peptide-encoding MC DNA, but not the corresponding full-length (FL) pDNA, conferred significant protection in mice challenged with Listeria monocytogenes expressing the model peptide. Together, our results suggest intradermal delivery of MC DNA may prove more efficacious for prophylaxis than traditional pDNA vaccines.

Development of Poly(β-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.

Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation.