Polyguanylic Acid Research Articles

The inhibition of MARCO by PolyG alleviates pulmonary fibrosis via regulating mitochondrial function in a silicotic rat model.

Silicon dioxide (SiO2)-induced pulmonary fibrosis is potentially associated with the impairment of mitochondrial function. Previous research found that inhibition of macrophage receptor with collagenous structure (MARCO) could alleviate particle-induced lung injury by regulating phagocytosis and mitigating mitochondrial damage. The present study aims to explore the underlying anti-fibrosis mechanism of polyguanylic acid (PolyG, MARCO inhibitor) in a silicotic rat model. Hematoxylin and eosin and Masson staining were performed to visualize lung tissue pathological changes. Confocal microscopy, transmission electron microscope, western blot analysis, quantitative real-time PCR (qPCR), andadenosine triphosphate (ATP) content assay were performed to evaluate collagen content, mitochondrial function, and morphology changes in SiO2-induced rat pulmonary fibrosis. The results suggested that SiO2 exposure contributed to reactive oxygen species aggregation and the reduction of respiratory complexes and ATP synthesis. PolyG treatment could effectively reduce MARCO expression and ameliorate lung injury and fibrosis by rectifying the imbalance of mitochondrial respiration and energy synthesis. Furthermore, PolyG could maintain mitochondrial homeostasis by promoting peroxisome proliferator-activated receptor-coactivator 1 α (PGC1α)-mediated mitochondrial biogenesis and regulating fusion and fission. Together, PolyG could ameliorate SiO2-induced pulmonary fibrosis via inhibiting MARCO to protect mitochondrial function.

Cationic porphyrin TMPyP4 redox behaviour and interaction with nucleic acids: Towards a new methodology for screening porphyrin-based anticancer drugs

The cationic meso-substituted porphyrin 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP4) is considered an excellent model compound for the development of new porphyrin-based anticancer drugs able to inhibit the telomerase in cancer cells. Drug development testing methods are nowadays laborious and time-consuming; hence, the development of rapid, sensitive, selective, low-cost techniques of testing the effectiveness of drugs acting on specific DNA sequences is essential. TMPyP4 oxidation behavior and its interaction with different nucleic acid sequences were electrochemically investigated, aiming to develop an easy, label free and low cost electrochemical methodology of fast screening porphyrin-based anticancer drugs, by differentiating their affinity to different nucleic acid structures.TMPyP4 undergoes an irreversible, pH dependent, two-step oxidation at glassy carbon electrode, each step occurring with the loss of one electron and one proton from the nitrogen atoms of the porphine central ring. DPV was able to discriminate between TMPyP4 binding to different nucleic acid sequences, in agreement with models existent in the literature: (i) TMPyP4 interaction with DNA double-helix is fast and occurs by intercalation, preferentially at the G-C base pairs, but does not cause oxidative DNA damage, (ii) TMPyP4 interaction with polyadenylic acid single-helices formed at neutral pH occurs by pseudo-intercalation, and with single- and double-helices formed at mild acidic pH occurs by side-binding, and (iii) TMPyP4 interaction with polyguanylic acid promotes the formation of G-quadruplexes. The voltammetric results represent a proof of concept, confirming the ability of the proposed electrochemical methodology to screen porphyrin-based anticancer drugs in the process of drug development, allowing the drug easy classification in terms of intercalation and selectivity for different nucleic acid structures.

Development of Polynucleotide-loaded Nanoparticles for the Regulation of Intracellular Nucleotide Levels

Polynucleotide-loaded nanoparticles are potentially useful for the regulation of intracellular nucleotide levels. Polyadenylic acid and polyguanylic acid were used as model compounds for encapsulation into poly(lactic-co-glycolic acid) nanoparticles. The properties of the produced nanoparticles, as well as the effect of the polynucleotide molecular size on encapsulation into the nanoparticles were investigated.

PolyG mitigates silica-induced pulmonary fibrosis by inhibiting nucleolin and regulating DNA damage repair pathway

Polyguanylic acid potassium salt (PolyG) has an anti-fibrotic G-quadruplex (G4) structure. It could inhibit the expression of nucleolin, a protein involved in cell proliferation and apoptosis. However, its role in regulating nucleolin in silicosis is still unknown. After instillation of 50 μl of crystalline silica suspension (50 mg/ml) into the trachea of C57BL/6 mice, we show that nucleolin expression is upregulated in mouse pulmonary tissue following the treatment with silica and that PolyG, which were injected 2.5 mg/kg body weight into mice by abdomen, could alleviate pulmonary fibrosis through inhibiting the expression of nucleolin. Further, we demonstrated that the expression of the DNA double-strand break (DSB) marker, γ-H2AX, increased in response to silica treatment. PolyG could efficiently reduce the protein expression of γ-H2AX and decreased the level of fibrosis-related genes, such as Col1a1 and Col3a1, as well as the levels of fibrosis-associated proteins α-SMA and vimentin in the lungs of silica-treated mice. These findings show that PolyG could regulate nucleolin and DNA damage repair to control fibrotic response in experimental silicosis and provide a new target for preventive intervention.

Internalization of scavenger receptor ligands by cortical neurons.

Scavenger receptors (SRs) are a family of receptors displaying affinity for a wide variety of ligands including modified lipoproteins. SRs may play a range of physiological functions including intracellular transport, lipid transport and pathogen clearance. The role of SRs has been documented in pathologies such as atherosclerosis and Alzheimer's disease. Although most studies on SRs have focused on macrophages, they are also present in other cells like endothelium, smooth muscles and brain tissue. Within brain, due to its functional similarity, SRs have been studied mostly in microglia. However, in situ images from Allen's brain atlas suggest SRs are abundant in neurons. In this study we have used two fluorophore labeled well characterized SR ligand, maleylated-BSA (MBSA) and polyguanylic acid (poloyG) to probe acute cortical slices. Our data indicate that within cortex, neurons avidly endocytose both ligands. Thus in cerebral cortex neurons may have higher number of functional SRs on the surface than other cell-types.

Atomic Force Microscopy and Voltammetric Investigation of Quadruplex Formation between a Triazole-Acridine Conjugate and Guanine-Containing Repeat DNA Sequences.

The interactions of the Tetrahymena telomeric repeat sequence d(TG4T) and the polyguanylic acid (poly(G)) sequence with the quadruplex-targeting triazole-linked acridine ligand GL15 were investigated using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite and voltammetry at a glassy carbon electrode. GL15 interacted with both sequences, in a time dependent manner, and G-quadruplex formation was detected. AFM showed the adsorption of quadruplexes as small d(TG4T) and poly(G) spherical aggregates and large quadruplex-based poly(G) assemblies, and voltammetry showed the decrease and disappearance of GL15 and guanine oxidation peak currents and appearance of the G-quadruplex oxidation peak. The GL15 molecule strongly stabilized and accelerated G-quadruplex formation in both Na(+) and K(+) ion-containing solution, although only K(+) promoted the formation of perfectly aligned tetra-molecular G-quadruplexes. The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the G-quadruplex complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string.

Binding affinities of folic acid and related pterins with biological macromolecules under physiological conditions

Abstract Folic acid and pterin derivatives are important heterocyclic compounds found in a variety of biological systems and have been shown to be photochemically active. Understanding the amount of binding that various pterins have with biological macromolecules under physiological conditions is important in predicting what specific biomolecules will bind with pterins and may, therefore, result in photochemical damage from charge-transfer reactions. The relative binding of folic acid, or pteroyl-L-glutamic acid (PteGlu), 6-methylpterin (Mep), 6-hydroxymethylpterin (Hmp), 6-formylpterin (Fop), and 6-carboxypterin (Cap) with bovine serum albumin (BSA), electrically neutral lipid (ENL), polyguanylic acid (Poly G), polycytidylic acid (Poly C), polyadenylic acid (Poly A), polythymidylic acid (Poly T), Micrococcus luteus DNA (72% GC), Escherichia coli DNA (50% GC), calf thymus DNA (42% GC), and Clostridium perfrigens DNA (27% GC) in neutral phosphate buffer were studied. Our results indicate that PteGlu demonstrated strong binding to neutral lipids, while the other pterins showed minimal binding, and BSA had a significant binding to PteGlu, Cap, and especially Fop. Our results also reveal a high affinity for DNA by PteGlu, which suggests that a relatively high percentage of folic acid is bound to DNA before photochemistry occurs.

Ethidium Bromide Interaction with Poly(G)

In current work we investigate the interaction between polyguanylic acid and Ethidium Bromide ( EtBr ) and the changes of thermodynamic parameters due to those interactions by spectrophotometric methods. From the binding isotherms, binding constants were calculated for three different temperatures and the changes of Gibbs free energy, entropy and enthalpy for complex-formation were determined. Complex-formation with four-stranded poly(G) structure needs less conformational changes in contrast with poly(G)poly(C), and due to that, EtBr binds with 5 and not 4 nucleotides. Obtained results confirm the concept that polyguanylic acids may form four-stranded complex structures with intercalating ligand EtBr in the aqueous solution.

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.

Time-dependent polyguanylic acid structural modifications

Polyguanylic acid (poly(G)) structural modifications are dependent on the presence of K+ or Na+ ions and incubation time, and were studied using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite (HOPG) and voltammetry at a glassy carbon electrode. AFM images showed the poly(G) single-strand self-assembling into short G-quadruplex (GQ) regions after low incubation time, while large GQ poly(G) morphology aggregates with low adsorption were formed after high incubation times, decreasing the HOPG surface coverage. The voltammetry in freshly prepared poly(G) solutions showed the occurrence of the guanine (G) residues oxidation peak in the poly(G) single-strand. Increasing the incubation time, the G oxidation peak decreased and disappeared, and a GQ residues oxidation peak in the GQ poly(G) morphology appeared, at a higher oxidation potential, dependent on the incubation time, presenting a maximum after 10days of incubation, and reaching a steady value after ~17days of incubation.

Raman Spectroscopic Study of Nucleobase Pairs and Mispairs - A Potential Method for Analysis of DNA Sequences

To exploit sensitive, accurate and convenient detection techniques for nucleic acid sequences can help to overcome the defects of current fluorescence detection method. Raman spectroscopy represents one family of methods that may offer such potential. In this paper, G•C base pairs and C•U mispairs were studied by Raman spectral characterization of single-strand homopolynucleotides, i.e. polyguanylic acid (PolyG), polycytidylic acid (PolyC) and polyuridylic acid (PolyU), and their equimolar complexes PolyG•PolyC and PolyC•PolyU in solution of 0.14 M Na+, pH7.0. Both of the complexes were found to adopt A type conformation. Helix formation between complementary polynucleotides led to marked and characterisctic changes in frequency and intensity of Raman bands of the biopolymers. Spectral changes were also observed upon the formation of C•U mispairs. According to the Raman data, in C•U mispairs the C2=O of U was engaged in a hydrogen bond to the N4H2group of cytosine, while the uracil C4=O group is free. Some of the rC residues transformed from C3-endo/anti to C2-endo/anti conformation due to base mispairing.The results suggest that Raman spectroscopy provides detailed and highly sensitive information about both the structure of nucleic acids and their base composition, and has great potential for analysis of DNA sequences.

Effect of Quadruplex Conformation on Radiation-Induced Formation of 8-Hydroxyguanine and Unaltered Base Release in Polyguanylic Acid

The ability of guanine-rich sequences to form quadruplex structures in telomeres for example is important in a number of biological processes such as aging, carcinogenesis and gene regulation. Ionizing radiation can cause damage to guanine moieties that can affect the stability or formation of the guanine quadruplex structures. In addition, the mechanisms of formation of these radiation damages in quadruplex structures may be different from those that occur in single- or double-stranded conformations. We have studied the quantitative aspects of the radiation induced formation of 8-hydroxy-2'-guanine base modifications and unaltered guanine base release in single-, double- and four-stranded conformations of polyriboguanylic acid as a model of guanine-rich sequences in telomere-like structures. The results show that the strandedness of guanine-rich sequences is an important variable in the observed yields of these base damages and suggests that telomere-like structures with G-quadruplexes will be relatively more radiosensitive than the other regions of duplex DNA. Hydroxyl radicals are the major reactive species that produce the DNA damage, although the presence of oxygen significantly reduces their radiation yields for all conformations of polyriboguanylic acid and changes the proportions of the yields of the various damages among the polymer conformations.

Inhibitor of REL/NF- КB is regulated in Sydney rock oysters in response to specific double-stranded RNA and Vibrio alginolyticus, but the major immune anti-oxidants EcSOD and Prx6 are non-inducible

Invertebrates, such as oysters have mechanisms to recognize different microbial associated molecular patterns ( MAMPs) and recognition of these antigens triggers signaling pathways for the transcription of immune-related proteins. Components of the Rel/NF- КB pathway have been identified in mollusks but the role of different MAMPs in the activation of this pathway is poorly understood. In the current study, the Sydney rock oyster ( Saccostrea glomerata) was injected intramuscularly with a range of different MAMPs. Oysters were sampled over a 24 h period and the difference in expression of genes associated with immunity was determined by quantitative reverse transcription polymerase chain reaction ( qRT-PCR) using RNA extracted from hemocytes. The results showed that oysters can discriminate between different nucleic acids and double-stranded RNA sequences for activation of the Rel/NF- КB pathway in S. glomerata. Polyinosinic–polycytidylic acid ( poly I:C) up-regulated expression of inhibitor of Rel/NF- КB ( I КB ) by 26-fold and interferon-inhibiting cytokine ( IK) by 2.2-fold, whereas polycytidylic acid–polyguanylic acid ( poly G:C) and double stranded Vibrio alginolyticus genomic DNA ( dsDNA) did not. I КB was also up-regulated 3.8-fold higher for oysters stimulated with V. alginolyticus bacterial cells suggesting that lipopolysaccharide ( LPS) also activates the Rel/NF- КB pathway. The expressions of extracellular superoxide dismutase ( EcSOD) and peroxiredoxin 6 ( Prx6) were not significantly affected by dsRNA, dsDNA or V. alginolyticus bacterial cells suggesting that these two genes, previously demonstrated to be important in the resistance of S. glomerata to disease are not directly inducible by these MAMPs. This is the first study investigating differences in expression kinetics of immune genes in response to challenge from different MAMPs in this economically important bivalve species.

A study of the pH dependence of electronically excited guanosine compounds by picosecond time-resolved infrared spectroscopy

The photophysical properties of 5'-guanosine monophosphate (5'-GMP) and polyguanylic acid {poly(G)} in D(2)O solutions of varying pH have been studied using picosecond transient infrared absorption spectroscopy. Whereas in neutral or weakly alkaline solution only the vibrationally excited electronic ground state of 5'-GMP is observed, in acidic solution the relatively long-lived (229 +/- 20 ps) electronic excited state of protonated 5'-GMP, which possesses strong absorptions at 1517 and 1634 cm(-1), could be detected. The picosecond transient behaviour of polyguanylic acid in acidic solution is also very different from that of the polynucleotide in neutral solution due not only to the protonation of guanine moieties yielding the protonated excited state but because of the disruption of the guanine stacks which are present in the species in neutral solution.

The Application of DNA-Biosensors and Differential Scanning Calorimetry to the Study of the DNA-Binding Agent Berenil.

The in situ DNA-damaging capacity of berenil (1) has been investigated usingan electrochemical approach employing double stranded (ds) DNA-modified glassy carbonelectrode biosensors. Electrochemical voltammetric sensing of damage caused by 1 todsDNA was monitored by the appearance of peaks diagnostic of the oxidation of guanineand adenine. When 1 was incorporated directly onto the biosensor surface, DNA damagecould be observed at concentrations of additive as low as 10 μM. In contrast, when thedsDNA-modified biosensor was exposed to 1, in acetate buffer solution, the method wasmuch less sensitive and DNA damage could be detected only in the presence of 100 μMberenil. When mixed solutions of 1 and single stranded (ss) DNA, polyguanylic acid orpolyadenylic acid were submitted to voltammetric study, the oxidation signals of therespective bases decreased in a concentration-dependent manner and the major variation ofthe adenine current peak indicated preferential binding of 1 to adenine. The electrochemical results were in close agreement with those deriving from a differentialscanning calorimetric study of the DNA-berenil complex.

Inhibition of Macrophage Phagocytotic Activity by a Receptor-targeted Polymer Vesicle-based Drug Delivery Formulation of Pravastatin

Ruptures of macrophage-rich atherosclerotic plaques in the coronary arteries are the main reason for heart attack. Targeted therapeutic interventions with an inhibitory effect on the macrophages promise to be beneficial, but currently available drugs such as statins achieve event reductions of only 30%. Dose-limiting adverse effects in remote organs prohibit achieving higher drug levels known to have strong inhibitory effects on macrophages. Receptor-specific targeting using statin-loaded nanometer-sized triblock copolymer vesicles with targeting moieties might allow high-dose treatment for improved efficacy, while minimizing toxicity in other cells. Vesicle uptake by target cells but not other cell types and slow intracellular content release was observed. A major improvement in biologic efficacy was observed for polymer vesicles compared to free drug, whereas no increased cytotoxicity was observed in muscle cells. Such high-dose, targeted therapy of statins through cell-specific polymer vesicles allows novel treatment paradigms not only for atherosclerosis, but appears promising for a wide range of drugs and diseases.

Raman spectroscopic study of G—A mismatches

G?A mismatches are non-canonical base pairs that widely occur in native nucleic acids. They have been found to be functionally important in adopting unusual structures. In this paper, G?A mispairing was studied by Raman spectral characterization of Polyadenylic acid (PolyA), Polyguanylic acid (PolyG) and their equimolar mixture in solution of 0.08 mol/L Na+, pH7.0. The experiment showed the following three results. (1) At the experimental conditions used in the present work, PolyA and G?A complexes existed as single-stranded and double-stranded helix of A form, respectively. And PolyG mainly occurred to be anti-parallel quadruplex conformers. (2) The formation of G?A complexes, on one hand strengthened parts of base stacking interactions especially for PolyG, leading to Raman hypochromism effect with some corresponding bands shifting, and on the other hand weakened other base stacking interactions especially for PolyA to a certain degree. During this process, the backbone of PolyG underwent a significant change, but PolyA still conserved its main chain conformation. (3) The formation of G?A complexes was stabilized by two interbase hydrogen-bond interactions (i.e. N6H2(A)?N3 (G) and N7 (A)?N2H2(G)) and a third hydrogen bond between O2' (G) and N6 (A). The third hydrogen bond was responsible for the remarkable changes of PolyG backbone conformation.

RNA targeting by small molecule alkaloids: Studies on the binding of berberine and palmatine to polyribonucleotides and comparison to ethidium

The binding affinity, energetics and conformational aspects of the interaction of isoquinoline alkaloids berberine and palmatine to four single stranded polyribonucleotides polyguanylic acid [poly(G)], polyinosinic acid [poly(I)], polycytidylic acid [poly(C)] and polyuridylic acid [poly(U)] were studied by absorption, fluorescence, isothermal titration calorimetry and circular dichroism spectroscopy and compared with ethidium. Berberine, palmatine and ethidium binds strongly with poly(G) and poly(I) with affinity in the order 10 5 M −1 while their binding to poly(C) and poly(U) were very weak or practically nil. The same conclusions have also emerged from isothermal titration calorimetric studies. The binding of all the three compounds to poly(C) and poly(I) was exothermic and favored by both negative enthalpy change and positive entropy change. Conformational change in the polymer associated with the binding was observed in poly(I) with all the three molecules and poly(U) with ethidium but not in poly(G) and poly(C) revealing differences in the orientation of the bound molecules in the hitherto different helical organization of these polymers. These fundamental results may be useful and serve as database for the development of futuristic RNA based small molecule therapeutics.

Picosecond infrared probing of the vibrational spectra of transients formed upon UV excitation of stacked G-tetrad structures

The photophysical properties of stacked G-tetrads in diverse systems, including concentrated solutions of 5'-guanosine monophosphate (5'-GMP), polyguanylic acid (poly(G)) and the G-rich oligodeoxynucleotide sequence characteristic of human telomeric DNA, are probed by ps-TRIR and compared to those of the monomeric 5'-GMP.

Evolution and Function of Leukocyte RNase A Ribonucleases of the Avian Species, Gallus gallus

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.