Mg2+-induced DNA compaction, condensation, and phase separation in gene delivery vehicles based on zwitterionic phospholipids: a dynamic light scattering and surface-enhanced Raman spectroscopic study.

Despite the significant efforts towards applying improved non-destructive and label-free measurements of biomolecular structures of lipid-based gene delivery vectors, little is achieved in terms of their structural relevance in gene transfections. Better understanding of structure-activity relationships of lipid-DNA complexes and their gene expression efficiencies thus becomes an essential issue. Raman scattering offers a complimentary measurement technique for following the structural transitions of both DNA and lipid vesicles employed for their transfer. This work describes the use of SERS coupled with light scattering approaches for deciphering the bioelectrochemical phase formations between nucleic acids and lipid vesicles within lipoplexes and their surface parameters that could influence both the uptake of non-viral gene carriers and the endocytic routes of interacting cells. As promising non-viral alternatives of currently employed risky viral systems or highly cytotoxic cationic liposomes, complexations of both nucleic acids and zwitterionic lipids in the presence of Mg2+ were studied applying colloidal Ag nanoparticles. It is shown that the results could be employed in further conformational characterizations of similar polyelectrolyte gene delivery systems.

Thermodynamics of molecular recognitions between antineoplastic drug taxol and phosphatidylcholine

The aim of this study was to study the basic features of Taxol recognition with phospholipids by applying the thermodynamic and spectroscopic measurements. The obtained information could be used further for deductions on its precise cellular and pharmacological mechanisms of action, on improvements of its solubility properties by phospholipids, as well as for designing the novel lipidic carriers for drug delivery.

AUF1, the regulator of tumor necrosis factor alpha messenger RNA decay, is targeted by autoantibodies of patients with systemic rheumatic diseases.

OBJECTIVE: To investigate which members of the heterogeneous nuclear RNP (hnRNP) family are targeted by autoantibodies from patients with systemic rheumatic diseases. METHODS: Using a semipurified preparation of natural hnRNP proteins, 365 sera from patients with rheumatic diseases and control subjects were screened by immunoblotting for the presence of autoantibodies. Bacterially expressed recombinant hnRNP D (AUF1) proteins were used for confirming the data obtained. Binding of RNA and autoantibody to AUF1 was investigated by gel retardation assays. Expression of AUF1 in cultivated cells and synovial tissue was analyzed by indirect immunofluorescence and immunohistochemistry. RESULTS: Autoantibodies to AUF1 proteins were detected in 33% of patients with systemic lupus erythematosus, 20% of patients with rheumatoid arthritis, 17% of patients with mixed connective tissue disease, and <10% of patients with other rheumatic disorders. Epitope mapping studies showed the autoantibodies to be directed to conformational epitopes in the N-terminal RNA-binding part of AUF1. However, autoantibody binding did not interfere with RNA binding as assessed by gel-shift assays. Immunohistochemical studies revealed AUF1 to be expressed in the cytoplasm of RA synovial tissue as compared with nuclear staining in osteoarthritis and normal synovium, particularly in macrophages of the lining layer and in fibroblasts of the sublining areas. CONCLUSION: These data identify AUF1 proteins as novel autoantigens in SLE and related autoimmune disorders. Because AUF1 proteins are major components of messenger RNA stability complexes, our findings suggest that these complexes form a novel macromolecular target structure for autoantibodies in rheumatic autoimmune diseases.

Nucleic acid-phospholipid recognition: Fourier transform infrared spectrometric characterization of ternary phospholipid-inorganic cation-DNA complex and its relevance to chemicopharmaceutical design of nanometric liposome based gene delivery formulations.

The present work is a continuation of our previous microscopic, spectroscopic and microcalorimetric measurements of liposomes and poly(ribo)nucleotides and their ternary complexes with inorganic cations as an alternative formulation employing zwitterionic phospholipids instead of cytotoxic cationic lipids. Current report describes Fourier transform infrared spectrometric study as employed to follow structural transitions of newly proposed ternary solid neutral lipid-Mg(2+)-DNA complexes as promising gene delivery formulation. Spectra of the unbound components are compared with those obtained after their complexation as binary and ternary mixtures. Results are described at the levels of carbonyl, phosphate, choline and CH groups and discussed as effects of nucleic acid and phosphatidylcholine moiety on each other in the absence and in the presence of Mg(2+). The infrared spectra of DNA-lipid phases are dominated by the lipid specific absorption bands, with a very little contribution of DNA. Data suggest that upon recognition of DNA with lipids, the DNA undergoes helical transition. Mg(2+) effects are interpreted as dehydrations of phosphates and H-bonding inducing effects on carbonyl groups. The role of residual and surface water on these associations, as well as on chain packing is also discussed followed by possible implications of the ternary complex formation for further gene transfer designs.

Phospholipid-nucleic acid recognition: energetics of DNA-Mg2+-phosphatidylcholine ternary complex formation and its further compaction as a gene delivery formulation.

Thermodynamic features related to the preparation and use of self-assemblies formed between multilamellar and unilamellar zwitterionic liposomes and polynucleotides with various conformation and sizes are presented. The divalent metal cation-induced adsorption, aggregation, and adhesion between single- and double-stranded polyribonucleotides and phosphatidylcholine vesicles was followed by differential adiabatic scanning microcalorimetry. Nucleic acid condensation and compaction mediated by Mg2+ was followed, with regard to interfacial interaction with unilamellar vesicles. Microcalorimetric measurements of synthetic phospholipid vesicles and poly(ribo)nucleotides and their ternary complexes with inorganic cations were used to build the thermodynamic model of their structural transitions. The increased thermal stability of the phospholipid bilayers is achieved by affecting their melting transition temperature by nucleic acid-induced electrostatic charge screening. Measurements give evidence for the stabilization of polynucleotide helices upon their association with liposomes in the presence of divalent metal cations. Such an induced aggregation of vesicles leads either to heterogeneous multilamellar DNA-lipid arrangements or to DNA-induced bilayer destabilization and lipid fusion. The further employment of these polyelectrolyte nanostructures as improved formulations in therapeutic gene delivery trials, as well as in DNA chromatography, is discussed.

Phospholipid-nucleic acid recognition: developing an immobilized liposome chromatography for DNA separation and analysis.

The basic physicochemical principles that determine nucleic acid-phospholipid recognition and self-assembly are presented, focusing on their use as promising stationary phases for separation and analyses of nucleic acids with various structures and properties. The utilization of immobilized liposome chromatography was designed as an aqueous, two-phase system for further studies of sequence- and topology-specific and non-specific nucleic acid binding features of phospholipids in the presence of cationic co-solutes. Such covalently attached liposomes are evaluated for their stability, binding affinities with nucleic acids and plasmids, as well as their subsequent compaction, in terms of their relevance to chromatographic applications.

Phospholipid-Nucleic Acid Complexation: Biomolecular Energetics of DNA-Mg(2+)-Phosphatidylcholine Ternary Complex Formation, Compaction and Relevance as Lipoplex Formulation.

Thermodynamic features related to preparation and use of self-assemblies formed between multilamellar and unilamellar zwitterionic liposomes and polynucleotides with various conformation and sizes are presented. The divalent metal cation induced adsorption, aggregation and adhesion between single- and double-stranded polyribonucleotides and phosphatidylcholine vesicles was followed by differential adiabatic scanning microcalorimetry. Nucleic acid condensation and compaction mediated by Mg(2+) was followed, with regard to interfacial interaction with unilamellar vesicles. Microcalorimetric measurements of synthetic phospholipid vesicles and poly(ribo) nucleotides and their ternary complexes with inorganic cations were used to build the thermodynamic model of their structural transitions. The increased thermal stability of the phospholipid bilayers is achieved by affecting their melting transition temperature by nucleic acid induced electrostatic charge screening. Measurements give evidence for the stabilization of polynucleotide helices upon their association with liposomes in presence of divalent metal cations. Such an induced aggregation vesicles either leads to heterogeneous multilamellar DNA-lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. The further employment of these polyelectrolyte nanostructures as an improved formulations in therapeutic gene delivery trials, as well as in DNA chromatography is discussed.

Preparation and phase behaviour of surface-active pharmaceuticals: self-assembly of DNA and surfactants with membranes. Differential adiabatic scanning microcalorimetric study.

Some energetics issues relevant to preparation and surface characterization of zwitterionic phospholipid-DNA self-assemblies, as alternative models of the currently used problematic lipoplexes are presented. Nucleic acid compaction capacities of Mg(2+) and N-alkyl-N,N,N-trimetylammonium ions (C(n)TMA, n=12) were compared, with regard to surface interaction with unilamellar vesicles. Differential adiabatic scanning microcalorimetric measurements of synthetic phosphatidylcholine liposomes and calf thymus DNA and their ternary complexes with Mg(2+) and C(12)TMA, were employed for deduction of the thermodynamic model describing their structural transitions. Small monodisperce and highly stable complexes are established after precompaction of DNA with detergent, followed by addition of liposomes. In contrast, divalent metal cation-mediated aggregation of vesicles either leads to heterogeneous multilamellar DNA-lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. Possible dependence of the cellular internalization and gene transfection efficiency on the structure and physicochemical properties of DNA-Mg(2+)-liposomes or DNA-cationic surfactant-liposome systems is emphasized by proposing the structure of their molecular self-organizations with further implications in gene transfer research.

Adiabatic differential scanning calorimetric study of divalent cation induced DNA - DPPC liposome formulation compacted for gene delivery

Complexes between nucleic acids and phospholipid vesicles have been developed as stable non-viral gene delivery vehicles. Currently employed approach uses positively charged lipid species and a helper zwitterionic lipid, the latter being applied for the stabilization of the whole complex. However, besides problematic steps during their preparation, cationic lipids are toxic for cells. The present work describes some energetic issues pertinent to preparation and use of neutral lipid-DNA self-assemblies, thus avoiding toxicity of lipoplexes. Differential scanning calorimetry data showed stabilization of polynucleotide helix upon its interaction with liposomes in the presence of divalent metal cations. It is thus possible to suggest this self-assembly as an improved formulation for use in gene delivery.

Divalent cation induced DNA-zwitterionic vesicle formulation compacted for gene delivery: thermodynamic aspects.

Complexes between nucleic acids and phospholipid vesicles have been developed as stable non-viral gene delivery vehicles. Currently employed approach uses positively charged lipid species and a helper zwitterionic lipid, the latter being applied for the stabilization of the whole complex. However, besides problematic steps during their preparation, cationic lipids are toxic for cells. Present work describes some energetics issues pertinent to preparation and use of neutral lipid-DNA self-assemblies, thus avoiding toxicity of lipoplexes. Differential scanning calorimetry data showed stabilization of polynucleotide helix upon its interaction with liposomes in the presence of divalent metal cations. It is thus possible to suggest this self-assembly as an improved formulation for use in gene delivery.

Molecular phylogenetics and functional evolution of major RNA recognition domains of recently cloned and characterized autoimmune RNA-binding particle.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are spliceosomal macromolecular assemblages and thus actively participate in pre-mRNA metabolism. They are composed of evolutionarily conserved and tandemly repeated motifs, where both RNA-binding and protein-protein recognition occur to achieve cellular activities. By yet unknown mechanisms, these ribonucleoprotein (RNP) particles are targeted by autoantibodies and hence play significant role in a variety of human systemic autoimmune diseases. This feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity. Since RNP domain is one of the most conserved and widespread scaffolds, evolutionary analyses of these RNA-binding domains can provide further clues on disease-specific epitope formation. The study presented herein represents a sequence comparison of RNA-recognition regions of recently cloned and characterized human hnRNP A3 with those of other relevant hnRNP A/B-type proteins. Their implications in human autoimmunity are particularly emphasized.