SEARCH · PubMed Health

Results for “Circular RNA”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources

PubMed1986

Dideoxy sequencing method using denatured plasmid templates.

The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved. The method is simple and rapid: the recombinant plasmid DNA is extracted and purified by rapid alkaline lysis followed by ribonuclease treatment. The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers. It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction. We examined each step of the procedure, and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA; (ii) a heptadecamer rather than a pentadecamer is recommended as a primer; and (iii) the sequencing reaction should be done at 37 degrees C or higher rather than at room temperature. The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA.

Base Sequence↗

PubMedDate not supplied

An improved method for the isolation of supercoiled DNA molecules using ion-exchange column chromatography.

An improved procedure is presented for the rapid isolation of supercoiled molecules using ion-exchange chromatography of alkali-denatured material. Under the conditions employed, contaminating cellular DNA, RNA, and nicked circular molecules are denaturable and are presented to the column as single strands while intact, covalently closed, supercoiled molecules resist denaturation and are batch eluted from the column as the only remaining duplex species. Small amounts of protein and short, duplex fragments (less than 1 kb) are removed in the void volume. There is no need for gradient fractionation. Supercoil fractions are virtually free of contamination with linear molecules as determined by electron microscopy, have an A260 nm/A280 nm ratio approaching 2.0, and are suitable for subsequent heteroduplex analysis, molecular cloning, subcloning, or restriction enzyme mapping. The method has been used successfully to purify plasmids up to 45 kb in length and to isolate rare circular replication intermediates from an overwhelming excess of contaminating linear molecules in retrovirus-infected cells.

Chromatography, Ion Exchange↗

PubMed2004

Studies on the compaction of isolated nucleoids from Escherichia coli.

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.

Chloramphenicol↗

PubMed2000

Decrease of wild-type and precore mutant duck hepatitis B virus replication during lamivudine treatment in white Pekin ducks infected with the viruses.

BACKGROUND/AIMS: Lamivudine, an antiviral agent, has been used in the treatment of chronic hepatitis B, but little is known about its effect on intrahepatic replication of hepatitis B virus. We investigated the effect of lamivudine on the replication of wild-type and precore mutant duck hepatitis B virus (DHBV) in the liver and serum of DHBV carrier ducks. METHODS: Chronic carrier ducks with either wild-type or precore mutant DHBV were treated for 2 weeks with either low-dose (20 mg/kg, p.o., b.i.d.) or high-dose lamivudine (100 mg/kg, p.o., b.i.d.) or were untreated. Serum levels of DHBV DNA were examined serially by slot-blot hybridization. A second group of chronic carrier ducks was treated for 12 weeks with lamivudine (100 mg/kg, p.o., b.i.d.) or was untreated. The amount of DHBV DNA in serum and its various replicative intermediates in the liver were serially examined by slot-, Southern, and Northern blot methods. RESULTS: In the 2-week treatment study, concentration of DHBV DNA in serum treated with low- and high-dose lamivudine was reduced to 10.8% and 1.1% of the control level in wild-type DHBV carriers, and to 2.3% and 0.48% in precore mutant DHBV carriers, respectively. In the 12-week treatment study, concentration of DHBV DNA in serum at the end of treatment was reduced to <0.65% and <5.36% in wild-type and precore mutant DHBV carriers, respectively. Southern and Northern blot analyses revealed that the various replicative forms of DHBV DNA in the liver were decreased in all treated ducks, but, covalently closed circular DNA and RNA intermediates tended to remain unchanged. CONCLUSIONS: Our results showed that lamivudine could reduce both wild-type and precore mutant DHBV levels in the liver through inhibition of the reverse transcription step, but complete elimination of the viruses from liver is difficult even by relatively long-term lamivudine monotherapy, suggesting a need for some additional therapy to obtain complete clearance.

Animals↗

PubMed1980

Sequence organization of feline leukemia virus DNA in infected cells.

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.

Animals↗

PubMed1990

Delta hepatitis. The disease and the virus.

Delta hepatitis virus is a new human pathogen always found associated with hepatitis B virus (HBV) causing both fulminant hepatitis and the accelerated progression of pre-existing HBV hepatitis. The virus is coated in HBsAg and contains circular single stranded RNA genome with very high intramolecular base-pairing, similar to the genomes of plant viroids, and the HDV antigen, a specific marker of HDV infection also found in the nuclei of infected hepatocytes. The genome is about 1700 nucleotides long and of minus polarity with a major constant antigenomic Open Reading Frame (ORF) which codes for the antigen. Replication of viral RNA appears to proceed by a rolling circle mechanism and specific self-cleavage and self-ligation of both genomic and antigenomic HDV RNA strands has been demonstrated in vitro. HDV appears to require HBsAg for its propagation and hepatotropism but not to depend on HBV for replication.

Animals↗

PubMed1997

Physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome.

A physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome was constructed. The macrorestriction map for CeuI, EagI, and SstII was created by ordering the 38 restriction sites by one- and two-dimensional pulsed-field gel electrophoresis (PFGE) and by using an original strategy based on the CeuI enzyme and indirect end labelling by hybridization on both sides of the CeuI sites with rrs (16S RNA) and 3' rrl (23S RNA) probes. The circular chromosome was estimated to be 4.15 Mb in size, and the average resolution of the physical map is 110 kb. The chromosome contains 11 rrn loci, which are localized on 44% of the chromosome in a divergent transcriptional orientation regarding the presumed location of the replication origin. In addition to these 11 rrn operons, a total of 40 identified genes were mapped by hybridization experiments with genes from C. acetobutylicum and from various other clostridia as probes. The genetic map of C. acetobutylicum was compared to that of the three other endospore-forming bacteria characterized so far: Bacillus subtilis, Clostridium beijerinckii, and Clostridium perfringens. Parodoxically, the chromosomal backbone of C. acetobutylicum showed more similarity to that of B. subtilis than to those of the clostridia.

Chromosomes, Bacterial↗

PubMed1996

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.

1-Deoxynojirimycin↗

PubMed2002

Energetic and conformational contributions to the stability of Okazaki fragments.

A combination of spectroscopic and calorimetric techniques was used to determine complete thermodynamic profiles accompanying the folding of a model Okazaki fragment with sequence 5'-r(gagga)d(ATCTTTG)-3'/5'-d(CAAAGATTCCTC)-3' and control DNA (with and without thymidine substitutions for uridine), RNA, and hybrid duplexes. Circular dichroism spectroscopy indicated that all DNA duplexes are in the B conformation, the RNA and hybrid duplexes are in the A conformation, and the Okazaki fragment exhibits a spectrum between the A and B conformations. Ultraviolet and differential scanning calorimetry melting experiments reveal that all duplexes unfold in two-state transitions with thermal stabilities that follow the order RNA > OKA > DNA (with thymidines) > hybrids > DNA (with uridines). The dependence of the transition temperature on salt concentration yielded counterion releases in the following order: DNA (with thymidines) > RNA > DNA (with uridines) > OKA > hybrids. Thus, Okazaki fragments have a conformation and charge density between those of its components DNA and hybrid segments. However, the presence of the RNA-DNA/DNA junction confers on them higher stabilities than their component hybrid and DNA segments. The binding of intercalators to an Okazaki hairpin of sequence 5'-r(gc)d(GCU5GCGC)-3' and to its control DNA hairpin has also been studied. The results show that the binding of intercalators to Okazaki fragments is accompanied with higher heats and lower binding affinities, compared with DNA duplexes. This suggests that the presence of an RNA/DNA junction yields a larger surface contact to interact with the phenanthroline ring of the intercalators, which may lead to a larger disruption of the flexible flanking bases of the junction. The overall results suggest that the presence of this junction stabilizes Okazaki fragments and provides a structural feature that can be exploited in the design of drugs to specifically target these molecules.

Calorimetry, Differential Scanning↗

PubMed2006

Characterisation of the RNA binding properties of the coronavirus infectious bronchitis virus nucleocapsid protein amino-terminal region.

The coronavirus nucleocapsid (N) protein binds viral RNA to form the ribonucleocapsid and regulate RNA synthesis. The interaction of N protein with viral RNA was investigated using circular dichroism and surface plasmon resonance. N protein underwent a conformational change upon binding viral RNA and the data indicated electrostatic interactions were involved in the binding of the protein to RNA. Kinetic analysis suggested the amino-terminal region facilitates long-range non-specific interactions between N protein and viral RNA, thus bringing the RNA into close proximity to N protein allowing specific contacts to form via a 'lure' and 'lock' mechanism.

Base Sequence↗

PubMed1973

DNA polymerase 3 star requires ATP to start synthesis on a primed DNA.

DNA polymerase III star replicates a varphiX174 single-stranded, circular DNA primed with a fragment of RNA. This reaction proceeds in two stages. In stage I, a complex is formed requiring DNA polymerase III star, ATP, spermidine, copolymerase III(*), and RNA-primed varphiX174 single-stranded, circular DNA. The complex, isolated by gel filtration, contains ADP and inorganic phosphate (the products of a specific ATP cleavage) as well as spermidine, polymerase III star, and copolymerase III star. In stage II, the chain grows upon addition of deoxynucleoside triphosphates; ADP and inorganic phosphate are discharged and chain elongation is resistant to antibody to copolymerase III star. Thus ATP and copolymerase III star are required to initiate chain growth but not to sustain it.

Adenosine Triphosphate↗

PubMed2026

mRNA therapy: A novel approach for retinal neurodegenerative diseases.

Retinal neurodegeneration remains a major cause of irreversible vision loss, yet current therapeutic options are limited in effectiveness. Although gene therapies have shown clinical potential, the overexpression platforms they rely on, such as adeno-associated virus DNA, are constrained by safety concerns, limited efficacy, and cargo size restrictions. In contrast, mRNA therapy has gained recognition as a compelling alternative, enabling rapid and efficient protein expression without the risk of genomic integration. This review synthesizes recent advances in mRNA engineering, delivery systems, and administration routes for retinal applications, and highlight strategies to enhance targeting, penetration, and controlled release through interdisciplinary collaboration between ophthalmology and bioengineering. In recent years, engineered mRNA formats, including chemically modified linear, circular, and self-amplifying RNA, can achieve higher translation efficiency within a tunable expression window. The transient nature and relatively low immunogenicity of in vitro transcribed mRNA support repeat dosing without insertional mutagenesis. Advances in nanocarriers, particularly lipid nanoparticles, have enabled preferential delivery to retinal neurons, Müller glia, and pigment epithelium via intraocular administration, while improving mRNA stability and transfection efficiency. In preclinical studies, mRNA has been widely used to deliver gene-editing tools, transcription factors, and supplementary functional proteins. In disease models such as optic nerve crush and laser-induced choroidal neovascularization, mRNA-based therapies enhance neuroprotection and suppress pathological angiogenesis in the injured retina, with favorable ocular safety profiles. However, it remains largely unexplored how the intrinsic advantages of mRNA therapy can be leveraged to develop tailored strategies for complex retinal disorders. Consistent with this gap, mRNA platforms have not yet been widely incorporated into retinal research or clinical practice. In parallel, clinical translation also lags: despite encouraging outcomes of lipid nanoparticle-mRNA formulations in preclinical models, no candidates have progressed into retinal clinical trials. This review draws on the complex pathology and therapeutic logic of retinal neurodegeneration. It proposes that mRNA therapy enables multitarget, repeatable, stage-specific interventions that align with the dynamic evolution of diseases and the requirements of combination therapy in retinal diseases. It may be used to support neuroprotection, axon regeneration, and neurovascular regulation. By integrating data across experimental models and modalities, this review outlines representative cases and experimental paradigms to guide rational trial design and carrier selection. Taken together, technical progress and evolving application strategies position mRNA therapy as a compelling therapeutic avenue for retinal neurodegeneration.

administration↗

PubMed2003

Replication of human hepatitis delta virus: recent developments.

In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). In hepatocytes infected with these two viruses, HDV RNA genomes are assembled using the envelope proteins of HBV. Since 1986, we have known that HDV has a small single-stranded RNA genome with a unique circular conformation that is replicated using a host RNA polymerase. These and other features make HDV and its replication unique, at least among agents that infect animals. This mini-review focuses on advances gained over the last 2-3 years, together with an evaluation of HDV questions that are either unsolved or not yet solved satisfactorily.

Gene Silencing↗

PubMed1997

The H3/H4 tetramer blocks transcript elongation by RNA polymerase II in vitro.

We have investigated transcript elongation efficiency by RNA polymerase II on chromatin templates in vitro. Circular plasmid DNAs bearing purified RNA polymerase II transcription complexes were assembled into nucleosomes using purified histones and transient exposure to high salt, followed by dilution and dialysis. This approach resulted in nucleosome assembly beginning immediately downstream of the transcription complexes. RNA polymerases on these nucleosomal templates could extend their 15- or 35-nucleotide nascent RNAs by only about 10 nucleotides in 15 min, even in the presence of elongation factors TFIIF and SII. Efficient transcript elongation did occur upon dissociation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arrested conformation when they encountered nucleosomes. Surprisingly, elongation was no more efficient on nucleosomal templates reconstituted only with H3/H4 tetramers, even in the presence of elongation factors and/or competitor DNA at high concentration. Thus, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.

Cell-Free System↗

PubMed1993

Using circular permutation analysis to redefine the R17 coat protein binding site.

The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used.

Bacteriophages↗

PubMed1985

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors.

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.

Base Sequence↗

PubMed2002

Cold denaturation of the hammerhead ribozyme.

Cold denaturation is a thermodynamic phenomenon resulting from a difference in the heat capacities, DeltaCp, of the folded and unfolded states of a macromolecule. Whereas this phenomenon has been extensively studied in proteins, it has been thought not to occur in nucleic acids due to a negligible DeltaCp of folding. Questioning the validity of this assumption, the low-temperature structure of the hammerhead ribozyme, a small catalytic RNA, was investigated by circular dichroism spectroscopy. In the presence of 10 mM Mg2+ at pH 5.0 and 40% methanol, a cold unfolding event likely corresponding to tertiary structure loss was observed with a Tm of -20 degrees C. In 500 mM NaCl at pH 6.6, and 40% methanol, large-scale unfolding of the ribozyme at both hot (Tm = 53 degrees C) and cold (Tm = -1 degrees C) temperatures occurred. Fitting of these data to a two-state model allowed determination of DeltaCp = 3.4 kJ mol-1 K-1, corresponding to >/=0.18 kJ K-1 (mol base pair)-1, in good agreement with recently published calorimetric values for DNA duplexes. These results constitute the first direct observation of cold denaturation of a nucleic acid, and point to the importance of DeltaCp terms in the thermodynamics of nucleic acid folding.

Base Sequence↗

PubMed1982

Comparative sequence and structure of viroid-like RNAs of two plant viruses.

A newly discovered group of spherical plant viruses contains a bipartite genome consisting of a single-strand linear RNA molecule (RNA 1, Mr 1.5 x 10(6) ), and a single-strand, covalently closed circular viroid-like RNA molecule (RNA 2, Mr approximately 125,000). The nucleotide sequences of the RNA 2 of two of these, velvet tobacco mottle virus and solanum nodiflorum mottle virus, have been determined. RNA 2 of solanum nodiflorum mottle virus consists of 377 residues whereas that of velvet tobacco mottle virus consists of two approximately equimolar species, one of 366 residues and the other, with a single nucleotide deletion, of 365 residues. There is 92-95% sequence homology between the RNA 2 species of the two viruses. The predicted secondary structures possess extensive intramolecular base pairing to give rod-like structures similar to those of viroids. The structural similarities between the RNAs 2 of velvet tobacco mottle virus and solanum nodiflorum mottle virus and viroids may reflect functional similarities.

Base Sequence↗