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Biophysical Journal May 2001This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic...
This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.
Topics: Cations; Cell Adhesion; Cell Membrane; Chromatography, High Pressure Liquid; DNA; Drug Delivery Systems; Ethanol; Freeze Fracturing; Humans; Light; Lipid Metabolism; Lipids; Liposomes; Magnetic Resonance Spectroscopy; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Models, Biological; Oligonucleotides, Antisense; Plasmids; Polyethylene Glycols; Polynucleotides; Protein Binding; Protein Structure, Tertiary; Pyrenes; Scattering, Radiation; Temperature; Ultracentrifugation
PubMed: 11325732
DOI: 10.1016/S0006-3495(01)76202-9 -
The Journal of General Physiology Jul 1966Polynucleotide kinase catalyzes the transfer of a phosphate group from ATP to the 5'-hydroxyl termini of polynucleotides. Selective labeling of the 5'-hydroxyl termini...
Polynucleotide kinase catalyzes the transfer of a phosphate group from ATP to the 5'-hydroxyl termini of polynucleotides. Selective labeling of the 5'-hydroxyl termini of DNA with polynucleotide kinase has been used to study the number and the identity of the 5'-terminal residues of bacteriophage DNA's, and to examine the nature of the phosphodiester bond cleavages produced by endonucleases and by sonic irradiation. The intact strands of T7 DNA bear 5'-phosphoryl end-groups; only deoxyadenylate and deoxythymidylate are present as 5'-terminal residues. The intact strands of native lambda-DNA bear 5'-hydroxyl end-groups. M13 DNA, a circular molecule, cannot be phosphorylated. End-group labeling of DNA provides a method for determination of molecular weight; calibration against other DNA preparations is not required. The molecular weight of a single strand of T7 DNA, determined by end-group labeling, is 13.1 x 10(6); the molecular weight of a single strand of lambda-DNA is 16.0 x 10(6). These values are in agreement with molecular weight estimates by sedimentation analysis and electron microscopy. Sonic irradiation of DNA has been shown to favor the production of polynucleotides terminated by 5'-phosphomonoester groups. All four deoxyribonucleotides are present as 5'-terminal residues of sonicated DNA.
Topics: Adenosine Triphosphate; Alkaline Phosphatase; Bacteriophages; Chemical Phenomena; Chemistry; Coliphages; DNA, Viral; Deoxyribonucleases; Escherichia coli; Molecular Weight; Phosphotransferases; Polynucleotides; Ultrasonics
PubMed: 5338564
DOI: 10.1085/jgp.49.6.81 -
Analytical Chemistry Oct 2012A polynucleotide probe, call a polymeric sequence probe (PSP), was used to detect influenza A (Influenza A/WSN/33) NA (Neuraminidase) viral RNA in Madin-Darby canine...
A polynucleotide probe, call a polymeric sequence probe (PSP), was used to detect influenza A (Influenza A/WSN/33) NA (Neuraminidase) viral RNA in Madin-Darby canine kidney (MDCK) cells. The PSP is a single-stranded DNA molecule with ~2,000 tandem repeat fluorescence binding sites and target binding sites that can bind with multiple fluorescence complementary oligos and target viral RNA using a fluorescence in situ hybridization (FISH) process. A single viral RNA labeled by PSP can be directly observed in MDCK cells. The simple FISH protocol enables the observation and quantitative analysis of the infectious process and drug effects with ultrahigh sensitivity and spatial resolution.
Topics: Animals; Dogs; In Situ Hybridization, Fluorescence; Influenza A virus; Madin Darby Canine Kidney Cells; Polynucleotides; RNA Probes; RNA, Viral
PubMed: 22978816
DOI: 10.1021/ac3023873 -
Nucleic Acids Research Feb 1977Ultraviolet light-induced free radical alkylation with 2-propanol or D-ribose, initiated with di-tert-butyl peroxide, of poly (G), poly (U20G), and poly(A) led to the...
Ultraviolet light-induced free radical alkylation with 2-propanol or D-ribose, initiated with di-tert-butyl peroxide, of poly (G), poly (U20G), and poly(A) led to the substitution of the appropriate group for the H-8 atom of the purines and addition across the 5,6-double bond of the pyrimidines. The alkylated polynucleotides were subjected to nucleolytic digestion with several nucleases. T1-RNase digestion of poly(G) irradiated with 2-propanol gave a mixture of the modified and non-modified mononucleotides. Similarly, pancreatic RNase digestion of the irradiated poly(U20G) resulted in a mixture of the appropriate mononucleotides. A T2-RNase treatment of poly(A) irradiated with 2-propanol gave the modified Ado-21:3'-P, while T2-RNase digestion of poly(A) irradiated with D-ribose led to the cyclic modified mononucleotides, in addition to the modified mononucleotides.
Topics: Alkylation; Free Radicals; Light; Poly A; Poly G; Polyribonucleotides; Ribonucleases
PubMed: 840644
DOI: 10.1093/nar/4.2.319 -
European Journal of Biochemistry Oct 1975A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer....
A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.
Topics: Bacteriophages; Enzyme Activation; Hydrogen-Ion Concentration; Kinetics; Manganese; Nucleotidyltransferases; Polynucleotides; Pseudomonas; Temperature; Templates, Genetic
PubMed: 241635
DOI: 10.1111/j.1432-1033.1975.tb02351.x -
Virology Journal Dec 2009Phosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro...
BACKGROUND
Phosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated.
RESULTS
In vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC50 of 0.045 microM and 0.16 microM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC50 of 0.05 microM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers.
CONCLUSION
These studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.
Topics: Administration, Oral; Animals; Antiviral Agents; Cells, Cultured; Cytomegalovirus Infections; DNA; Female; Humans; Mice; Mice, Inbred BALB C; Muromegalovirus; Poly C; Polynucleotides
PubMed: 19954538
DOI: 10.1186/1743-422X-6-214 -
Journal of Bacteriology Dec 1974A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical...
A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical isolates of Neisseria gonorrhoeae. The 70S ribosomes from sensitive strains were sensitive to the effects of Str and Spc on synthesis directed by several synthetic polynucleotide messengers, whereas 70S ribosomes from resistant strains were resistant to these same effects. In each case, the alteration was localized to the 30S ribosomal subunit by studying antibiotic sensitivities of hybrid 70S ribosomes formed by combining subunits from sensitive and resistant strains. No evidence was found for streptomycin- or spectinomycin-inactivating enzymes.
Topics: Amino Acids; Bacterial Proteins; Carbon Radioisotopes; Cell Fractionation; Cell-Free System; Drug Resistance, Microbial; Magnesium; Micropore Filters; Mutation; Neisseria gonorrhoeae; Polynucleotides; RNA, Messenger; Ribosomes; Spectinomycin; Streptomycin
PubMed: 4279906
DOI: 10.1128/jb.120.3.1293-1299.1974 -
Spectrochimica Acta. Part A, Molecular... Jan 2018The binding mode of thionine (3,7-diamino-5-phenothiazinium) with alternating and non-alternating DNA polynucleotides at low binding ratios was conclusively determined...
The binding mode of thionine (3,7-diamino-5-phenothiazinium) with alternating and non-alternating DNA polynucleotides at low binding ratios was conclusively determined using linear and circular dichroism spectroscopies. The binding to [poly(dG-dC)] and poly(dG)·poly(dC) was purely intercalative and was insensitive to ionic strength. Intercalative binding to [poly(dA-dT)] is observed at low ionic strength, but a shift of some dye to an non-intercalative mode is observed as the background salt concentration increases. With poly(dA)·poly(dT), intercalative binding is unfavourable, although some dye molecules may intercalate at low ionic strength, and groove binding is strongly promoted with increasing concentration of background salt. However, stacking with bases is observed with single-stranded poly(dA) and with triplex poly(dT)poly(dA)·poly(dT) which suggests that the unusual structure of poly(dA)·poly(dT) precludes intercalation. Thionine behaves similarly to the related dye methylene blue, and small differences may be attributed either to the ability of thionine to form H-bonds that stabilize intercalation or to its improved stacking interactions in the basepair pocket on steric grounds.
Topics: Absorption, Physicochemical; Circular Dichroism; DNA; Nucleic Acid Denaturation; Phenothiazines; Polynucleotides; Temperature
PubMed: 28800432
DOI: 10.1016/j.saa.2017.07.064 -
PloS One Mar 2011Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby...
Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.
Topics: Animals; Cell Line, Tumor; Mice; Molecular Structure; Polynucleotides; Ribosome Inactivating Proteins; Ricin; Shiga Toxin 2
PubMed: 21455295
DOI: 10.1371/journal.pone.0017883 -
Proceedings of the National Academy of... Jul 1971The sequential action of lambda exonuclease and polynucleotide ligase upon redundant joint molecules is sufficient to produce intact polynucleotide chains and...
The sequential action of lambda exonuclease and polynucleotide ligase upon redundant joint molecules is sufficient to produce intact polynucleotide chains and heat-stable, biologically active molecules of lambda DNA, whereas the action of ligase alone is insufficient. These results (a) confirm the previously described mechanism of single-strand assimilation, including a subsidiary mechanism by which the further action of lambda exonuclease is arrested when a redundant strand is completely assimilated, and (b) represent a simulation of the steps in genetic recombination that follow the formation of biparental complexes (synapsis). lambda exonuclease is postulated to catalyze a concerted reaction that includes exposure of complementary sequences, formation of heteroduplex regions, and elimination of redundant branches.
Topics: Biological Assay; Chromatography, DEAE-Cellulose; Coliphages; DNA, Viral; Deoxyribonucleases; Escherichia coli; Genetics, Microbial; Ligases; Phosphorus Isotopes; Polynucleotides; RNA, Transfer; Recombination, Genetic; Tritium
PubMed: 4934524
DOI: 10.1073/pnas.68.7.1639