ABSTRACT
In this work we report the design and synthesis of kinked oligonucleotide duplexes as potential inhibitors of HMGB1, a cytokine which triggers a broad range of immunological effects. We found that the designed ligands can interact with HMGB1, as evidenced by circular dichroism spectroscopy, and are able to block some extracellular effects induced by the protein, such as cellular proliferation and migration, as we demonstrated by in vitro biological assays. After selecting the most stable and active kinked duplex, we synthesized the corresponding PNA/DNA chimeric duplex which resulted to be more resistant to enzymatic degradation, and showed a biological activity comparable to that of the natural duplex. Preliminary in vivo assays in a mouse inflammatory model, showed a significant decrease of the mortality after administration of the PNA/DNA kinked duplex to LPS-treated mice.
Subject(s)
DNA/chemistry , HMGB1 Protein/chemistry , Nucleic Acid Heteroduplexes/chemistry , Protein Structure, Tertiary , Animals , Base Sequence , Binding Sites , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , DNA/metabolism , DNA/pharmacology , DNA, Circular , Endotoxemia/chemically induced , Endotoxemia/mortality , Endotoxemia/prevention & control , HMGB1 Protein/antagonists & inhibitors , HMGB1 Protein/metabolism , Lipopolysaccharides , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/metabolism , Nucleic Acid Heteroduplexes/pharmacology , Peptide Nucleic Acids/chemistry , Protein Binding , Survival Rate , Time FactorsABSTRACT
In this work, we report thermodynamic, kinetic, and microrheological studies relative to the formation of PNA- and PNA/DNA-based noncovalent polymeric systems, useful tools for biotechnological and bioengineering applications. We realized two kinds of systems: a PNA-based system formed by a self-assembling PNA tridendron, and a PNA/DNA hybrid system formed by a PNA tridendron and a DNA linker. The formation of a three-dimensional polymeric network, by means of specific Watson-Crick base pairing, was investigated by a detailed UV and CD spectroscopic study. Preliminary microrheology experiments were performed on both systems to evaluate their viscoelastic properties which resulted in agreement with the formation of soluble hyperbranched polymers that could be useful for drug/gene delivery, as well as for encapsulating organic pollutants of different shapes and sizes in environmental applications.
Subject(s)
DNA/chemistry , Peptide Nucleic Acids/chemistry , Polymers/chemistry , Base Pairing , Circular Dichroism , Dendrimers/chemistry , Kinetics , Rheology , Spectrophotometry, Ultraviolet , ThermodynamicsABSTRACT
In this work we report a kinetic and thermodynamic study relative to the formation of gel systems based on PNA and PNA/DNA dendrimers, useful for drug delivery or diagnostic applications. We realized two kinds of systems: a PNA-based monomolecular system formed by an autoassembling PNA tridendron (A) and a PNA/DNA bimolecular system based on a PNA tridendron with a mixed sequence and a DNA crosslinker (B). Both systems have the ability to form a three-dimensional network by means of specific W-C base pairing.
Subject(s)
DNA/chemistry , Dendrimers/chemistry , Peptide Nucleic Acids/chemistry , Base Pairing , Base Sequence , Cross-Linking Reagents , DNA/chemical synthesis , DNA/radiation effects , Dendrimers/chemical synthesis , Dendrimers/radiation effects , Gels , Kinetics , Molecular Conformation/radiation effects , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Peptide Nucleic Acids/chemical synthesis , Peptide Nucleic Acids/radiation effects , Photochemistry , Thermodynamics , Ultraviolet RaysABSTRACT
In continuing our research efforts for developing new oligodeoxynucleotide (ODN)-like drugs and diagnostics, we designed diaminobutyric peptide nucleic acids (dabPNAs), nucleopeptides characterized by a diaminobutyric-based building block that is an isomer of the aminoethylglycyl PNA (aegPNA) unit and the acyclic modification of the aminoprolyl PNA (ampPNA) monomer. In this work we present the solid phase synthesis of a dabPNA oligomer and of two aegPNAs containing a single dabPNA unit. A study relative to their binding ability towards DNA is also reported even in comparison with the well known aegPNAs.
Subject(s)
Butyric Acid/chemistry , DNA/chemistry , Glycine/analogs & derivatives , Peptide Nucleic Acids/chemical synthesis , Drug Design , Glycine/chemistry , Peptide Nucleic Acids/chemistryABSTRACT
Targeting regulatory RNA regions to interfere with the biosynthesis of a protein is an intriguing alternative to targeting a protein itself. Regulatory regions are often unique in sequence and/or structure and, thus, ideally suited for specific recognition with a low risk of undesired side effects. Targeting regulatory RNA elements, however, is complicated by their complex three-dimensional structure, which poses kinetic and thermodynamic constraints to the recognition by a complementary oligonucleotide. Oligonucleotide mimics, which shift the thermodynamic equilibrium towards complex formation and yield stable complexes with a target RNA, can overcome this problem. Peptide nucleic acids (PNA) represent such a promising class of molecules. PNA are very stable, non-ionic compounds and they are not sensitive to enzymatic degradation. Yet, PNA form specific base pairs with a target sequence. We have designed, synthesised and characterised PNA able to enter infected cells and to bind specifically to a control region of the genomic RNA of coxsackievirus B3 (CVB3), which is an important human pathogen. The results obtained by studying the interaction of such PNA with their RNA target, the entrance into the cell and the viral inhibition are herein presented.
Subject(s)
Antiviral Agents/pharmacology , Cytopathogenic Effect, Viral/drug effects , Enterovirus B, Human/drug effects , Peptide Nucleic Acids/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Proliferation/drug effects , Cells, Cultured , Coxsackievirus Infections/virology , Dose-Response Relationship, Drug , Enterovirus B, Human/chemistry , Enterovirus B, Human/genetics , HeLa Cells , Humans , Microbial Sensitivity Tests , Peptide Nucleic Acids/chemical synthesis , Peptide Nucleic Acids/chemistry , RNA, Viral/chemistry , RNA, Viral/drug effects , RNA, Viral/genetics , Structure-Activity RelationshipABSTRACT
A new synthetic strategy to get the PNA-3'DNA linker with the monomethoxytrityl (Mmt) group as temporary protection of the backbone to be used for the synthesis of PNA/DNA chimeras was employed and a convenient strategy to obtain Mmt PNA monomers was developed. The synthetic strategies take advantage of the introduction of the acid-labile Mmt-protecting group in the first step.
