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Interferon induction by two 2'-modified double-helical RNAs, poly(2'-fluoro-2'-deoxyinosinic acid) x poly(cytidylic acid) and poly(2'-chloro-2'-deoxyinosinic acid) x poly(cytidylic acid).

In addition to the 2'-azido analogue of (I)n x (C)n, (dIn3)n x (C)n, we have found two other (I)n x (C)n analogues, (dIfl)n x (C)n and (dIcl)n x (C)n, in which the 2'-hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon-inducing activity noted for various other 2'-halogeno analogues of (I)n x (C)n and (A)n x (U)n, i.e. (I)n x (dCcl)n, (dAfl)n x (U)n, (dAcl)n x (U)n, (A)n x (dUfl)n and (A)n x (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon-primed mouse L-929 cells, and intact rabbits, (dIfl)n x (C)n and (dIcl)n x (C)n induced interferon levels that were comparable to those induced by (I)n x (C)n. There was one particular system (L-929 cells treated with DEAE-dextran), however, in which (dIfl)n x (C)n and (dIcl)n x (C)n, unlike (I)n x (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n x (C)n and, to a lesser extent, (dIcl)n x (C)n were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n x (C)n. In their reactivity towards antibodies to double-stranded RNA (dIfl)n x (C)n and (dIcl)n x (C)n conformed more closely to (I)n x (C)n than did other 2'-substituted (e.g. 2'-O-methyl or 2'-O-ethyl) analogues of (I)n x (C)n. The high interferon-inducing potency of (dIfl)n x (C)n and (dIcl)n x (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n x (C)n and (dIcl)n x (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon-inducing capacity, and possibly other biological functions of double-stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2'-hydroxyl group.

Animals↗

Poly(rA) binds poly(rG).poly(rC) to form a triple helix.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix. Evidence for this structure includes ultraviolet absorbance mixing curves and melting curves, and circular dichroism spectroscopy. The formation of the triple helix depends on the length of the poly(rC) strand. Triple helix forms when the average length is around 100 nucleotides but does not form when the average length is about 500 nucleotides.

Circular Dichroism↗

The in vitro hydrolysis of poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] and poly(ethylene glycol).

This paper reports the study of the complete degradation process for a series of newly synthesized multi-block poly(ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] (PHB) as hard and hydrophobic block and poly(ethylene glycol) (PEG) as soft and hydrophilic segment. The initial stages of degradation of the poly(PHB/PEG urethane)s were monitored by carrying out the degradation experiments at pH 7.4 and 37 degrees C. The weight loss of the copolymer films was traced, and the degraded copolymer films were characterized by GPC, (1)H NMR, TGA, and SEM. The induction phase of the polymer degradation was characterized by a random chain scission of the ester backbone bonds of the PHB segments and an insignificant decline in the weight of the polymer films. An accelerated degradation process was carried out at pH 11.5 and 37 degrees C to investigate the long-term degradation behaviour. The characterization of the degraded polymer films was similar to that for the experiment at pH 7.4. In addition, the water-soluble degradation products were characterized by GPC, (1)H NMR, and FTIR. The main components of the water-soluble degradation products were found to be PEG blocks (monomeric up to quadmeric), 3-hydroxybutyric acid, and crotonic acid. It was found that the copolymer incorporating the highest amount of PEG degraded at the highest rate of all the copolymers studied. The complete degradation of the poly(PHB/PEG urethane)s was monitored using a combination of the physiological and accelerated hydrolytic degradation.

Biocompatible Materials↗

Study of the miscibility of poly(styrene-co-4-vinylbenzoic acid) with poly(ethyl methacrylate) or with poly[ethyl methacrylate-co-(2-N,N-dimethylaminoethyl) methacrylate] by inverse gas chromatography.

Poly(styrene) is immiscible with poly(ethyl methacrylate). The introduction of a small amount of 4-vinylbenzoic acid units along poly(styrene) chains (PS-VBA) enhanced its miscibility with poly(ethyl methacrylate) (PEMA) or with poly[ethyl methacrylate-co-(2-N,N-dimethylaminoethyl) methacrylate] (PEMA-DAE), as observed from the appearance of a single composition dependent glass transition temperature for each binary system using inverse gas chromatography. The negative values of the apparent polymer-polymer interaction parameter, chi(23)app, determined with different families of molecular probes, for three blend compositions and over a range of temperature confirm quantitatively the miscibility of these blends. The chi(23)app values for PEMA(PS-VBA) and (PEMA-DAE)-(PS-VBA) blends are dependent of the chemical nature of the probes, the temperature and the blend composition.

Chromatography, Gas↗

pH effect of coagulation bath on the characteristics of poly(acrylic acid)-grafted and poly(4-vinylpyridine)-grafted poly(vinylidene fluoride) microfiltration membranes.

The poly(acrylic acid)-graft-poly(vinylidene fluoride) (PAAc-g-PVDF) and poly(4-vinylpyridine)-graft-poly(vinylidene fluoride) (P4VP-g-PVDF) copolymers were obtained by thermally induced molecular graft copolymerization of acrylic acid (AAc) and 4-vinylpyridine (4VP), respectively, with the ozone-pretreated poly(vinylidene fluoride) (PVDF) in N-methyl-2-pyrrolidone (NMP) solution. Microfiltration (MF) membranes were prepared from the respective copolymers by phase inversion in aqueous media. The effects of pH of the coagulation bath on the physicochemical and morphological characteristics of the membranes were investigated. The surface compositions of the membranes were determined by X-ray photoelectron spectroscopy (XPS). The surface graft concentration of the AAc polymer for the PAAc-g-PVDF MF membrane increased with decreasing pH value of the coagulation bath. Completely opposite pH-dependent behavior was observed for the surface graft concentration of the 4VP polymer in the P4VP-g-PVDF MF membranes. A substantial increase in mean pore size was observed for the PAAc-g-PVDF MF membranes cast in basic coagulation baths of increasing pH. In the case of the P4VP-g-PVDF MF membranes, a substantial increase in mean pore size was observed for membranes cast in low pH (acidic) baths. The permeation rate of aqueous solutions through the PAAc-g-PVDF and P4VP-g-PVDF MF membranes exhibited a reversible dependence on the pH of the solution, with the membranes cast near the neutral pH exhibiting the highest sensitivity to changes in permeate pH.

Acrylates↗

Thermoresponsive release from poly(Glu(OMe))-block-poly(Sar) microcapsules with surface-grafting of poly(N-isopropylacrylamide).

Thermoresponsive microcapsules were prepared by grafting poly(N-isopropylacrylamide) (PNIPAAm) on the surface of polypeptide (poly(Glu(OMe))-block-poly(Sar) microcapsules. Naked poly(Glu(OMe)-block-poly(Sar) microcapsules were partly hydrolysed with NaOH to remove methyl groups and newly formed carboxyl groups were used to anchor polyallylamine having 4,4'-azobis(4-cyanovaleric acid) groups. Graft polymerization of N-isopropylacrylamide at the microcapsule surface was initiated by photo-cleavage of the azo groups. Microscopic examination showed that a homogeneous dense skin layer of PNIPAAm was formed on the surface of microcapsule at 40 degrees c, while the skin layer became loose when the temperature was lowered to 25 degrees C. Dextran release from the microcapsule was faster below the lower critical solution temperature (LCST) of PNIPAAm than that above it. When the temperature changed across the LCST, a reversible, thermoresponsive release from the microcapsule was observed. Notable, the transition of the release rate by changing the temperature occurs quickly in a narrow temperature range.

Acrylic Resins↗

Compatibilization effect of poly(epsilon-caprolactone)-b-poly(ethylene glycol) block copolymers and phase morphology analysis in immiscible poly(lactide)/poly(epsilon-caprolactone) blends.

The miscibility and phase behavior of two stereoisomer forms of poly(lactide) (PLA: poly (L-lactide) (PLLA) and poly(DL-lactide) (PDLLA)) blends with poly(epsilon-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) and PCL-b-monomethoxy-PEG (PCL-b-MPEG) block copolymers have been investigated by differential scanning calorimetry (DSC). The DSC thermal behavior of both the blend systems revealed that PLA is miscible with the PEG segment phase of PCL-b-(M)PEG but is still immiscible with its PCL segment phase although PCL was block-copolymerized with PEG. On the basis of these results, PCL-b-PEG was added as a compatibilizer to PLA/PCL binary blends. The improvement in mechanical properties of PLA/PCL blends was achieved as anticipated upon the addition of PCL-b-PEG. In addition, atomic force microscopy (AFM) measurements have been performed in order to study the compositional synergism to be observed in mechanical tests. AFM observations of the morphological dependency on blend composition indicate that PLA/PCL blends are immiscible but compatible to some extent and that synergism of compatibilizing may be maximized in the compositional blend ratio before apparent phase separation and coarsening.

Calorimetry, Differential Scanning↗

Biophysical characterization of complexation of DNA with block copolymers of poly(2-dimethylaminoethyl) methacrylate, poly(ethylene oxide), and poly(propylene oxide).

The interactions of DNA (salmon testes) with two new cationic block copolymers made of poly(2-dimethylaminoethyl) methacrylate and poly(ethylene oxide), PEO-pDMAEMA, or poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), L92-pDMAEMA, were studied with the aim to understand their different in vitro transfection efficiencies when used as nonviral delivery vectors. PEO-pDMAEMA does not show surface activity while L92-pDMAEMA is as surface active as its parent Pluronic L92. Surface tension, titration microcalorimetry, ethidium bromide displacement, and zeta-potential measurements were carried out in phosphate buffers at pH 5 and 7. The association of L92-pDMAEMA with DNA was strongly exothermic at both pHs; the critical aggregation concentration (CAC) corresponded to a N/P ratio of 0.3, the maximum energy evolved was reached for N/P ratios of 0.82 and 1.27 at pH 5 and pH 7, respectively, and the saturation occurred for N/P ratios close to 2. The presence of L92 in the structure of this new block copolymer apparently did not modify the thermodynamic parameters of the interaction with DNA. In contrast, the interaction with PEO-pDMAEMA was significantly less exothermic, and CAC and saturation occurred for N/Ps equal to 0.43 and 1.37, respectively. The strong affinity of L92-pDMAEMA for DNA was reflected in its capacity to displace ethidium bromide and in the jump in the values of the zeta potential when N/P is near 1. Above the N/P ratio at which electroneutral polyplexes are formed, only at pH 5 an excess of L92-pDMAEMA is incorporated in the complexes, resulting in positively charged complexes. The profile of the zeta-potential values obtained for mixtures of L92-pDMAEMA with Pluronic P123 showed a shift to a lower N/P ratio, owing to an easier interaction of L92-pDMAEMA molecules with DNA in the presence of P123. Additionally, a visual inspection of the systems indicates that P123 contributes to stabilize/solubilize the DNA/cationic polymer aggregates, by avoiding the typical phase separation near the charge neutralization point. The information obtained can be particularly useful to optimize the conditions to form efficient polyplexes for gene delivery systems.

Animals↗

Transitions of poly(dI-dC), poly(dI-methyl5dC) and poly(dI-bromo5dC) among and within the B-, Z-, A- and X-DNA families of conformations.

It is shown, using circular dichroism spectroscopy, that poly(dI-dC) is capable to isomerize into both Z-DNA and A-DNA in concentrated NaCl + NiCl2 and trifluoroethanol solutions, respectively. This polynucleotide also undergoes a cooperative, two-state transition in ethanol into a structure which most probably is a canonical B-DNA. This implies that the conformation of poly(dI-dC) is unusual in low-salt aqueous solution. The canonical B-DNA is also adopted by poly(dI-methyl5dC) in trifluoroethanol while this polynucleotide adopts Z-DNA not only in NaCl + NiCl2 but also in the presence of MgCl2. Poly(dI-methyl5dC) partially adopts X-DNA in concentrated CsF and mainly ethanolic solutions. Poly(dI-bromo5dC) isomerizes into Z-DNA not only in concentrated NaCl even in the absence of NiCl2 but also in concentrated MgCl2. This polynucleotide transforms between two distinct variants of Z-DNA in ethanol or trifluoroethanol solutions.

Circular Dichroism↗

Interaction of rat poly(A)-binding protein with poly(A)- and non-poly(A) sequences is preferentially mediated by RNA recognition motifs 3+4.

Vasopressin (VP) mRNA and the non-coding BC200 RNA are sorted to neuronal dendrites. Among proteins interacting specifically with both RNAs is the multifunctional poly(A)-binding protein (PABP) consisting of four RNA recognition motifs (RRMs) and a C-terminal auxiliary domain. The protein/RNA interaction studies presented here reveal that PABPs association with VP- and BC200 RNA is exclusively mediated by RRMs 3+4. Quantitative binding studies with PABP deletion mutants demonstrate preferential binding of RRMs 3+4 even to poly(A)-homopolymers, while RRMs 1+2 exhibit a lower affinity for those sequences. An optimal interaction with both poly(A)- and non-poly(A) sequences is only achieved by full-size PABP.

Animals↗

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails.

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

Escherichia coli Proteins↗

[Study of poly(C) and poly(I)-poly(C) complexes with model phospholipid membranes by infrared spectroscopy].

The temperature dependence of poly(C) is shown by the infrared spectroscopy to be different for the free polynucleotide and for the polynucleotide in complexes with membranes. The intensity of stretching vibrations of C = 0 bond of poly(C) in the complex appears to be sensitive to the temperature. The intensity of this band is sharply decreased by increasing the temperature. This effect depends upon concentration of Mg2+-cations. Adsorption of poly(I)-poly(C) on the surface of vesicles from phosphatidylcholine results in the increase of the double helix.

Hot Temperature↗