ABSTRACT
The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA-DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA-DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.
Subject(s)
DNA/chemistry , Peptides/chemistry , RNA/chemistry , Animals , DNA/genetics , Dimerization , Humans , Peptides/genetics , Protein Binding , RNA/geneticsABSTRACT
A simple and rapid procedure whereby human genomic DNA can be purified in a PCR amplifiable form from whole blood is described. In a first step, human genomic DNA is hybridized in solution to a biotinylated peptide nucleic acid (PNA), which forms a high-affinity triplex with A7 sequence motifs in the target DNA. The complex is then captured onto paramagnetic streptavidin-coated particles, which are subsequently transferred directly into the PCR. The purification method effectively removes inhibitors of the PCR from as much as 500 microL of whole blood.
Subject(s)
DNA/isolation & purification , Nucleic Acids , Peptides , Polymerase Chain Reaction , Biotin , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 7 , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA/blood , Factor IX/genetics , Female , Histidine , Humans , Male , Nucleic Acid Hybridization , Repetitive Sequences, Nucleic Acid , X ChromosomeABSTRACT
An optimized automated PNA synthesis protocol is reported. Under optimal conditions the product yield of a test 17-mer PNA is approximately 90%. The average coupling yield is 99.4%. The synthesis strategy is Boc/Z and the deprotected amine is neutralized in situ. The monomers are added in molar excess to HATU and pre-activated for 60 s before delivery to the resin. The concentration of the activated monomers is 0.08 M during the couplings. Heteroselective solvation provides the highest coupling yields. Acetic anhydride is used as capping reagent followed by a piperidine wash. The protocol has been developed in a 5 mumol scale but is easily scaled up to 10-50 mumol scale syntheses on the automated synthesizer (ABI 433A).
Subject(s)
Nucleic Acids/chemical synthesis , Peptides/chemical synthesis , Biopolymers , Chromatography, High Pressure Liquid , Glycine/analogs & derivatives , Glycine/chemistry , Piperidines/chemistry , Purines/chemistry , Pyrimidinones/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.
Subject(s)
Anthraquinones/metabolism , DNA/metabolism , Nucleic Acids/metabolism , Peptides/metabolism , Anthraquinones/chemical synthesis , Deoxyribonucleases, Type II Site-Specific/metabolism , Light , Nucleic Acids/chemical synthesis , Peptides/chemical synthesisABSTRACT
Acryloyl, methacryloyl, N-vinylcarbamoyl, and methacryloyl-glycyl derivatives of the morphine antagonist (--)-alpha-5,9-dimethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomorphane have been synthesized. The different unsaturated derivatives were copolymerized with appropriate comonomers to yield water-soluble polymers. Pharmacological tests in mice showed a 2--30 fold increase in duration of action in comparison to the free antagonist. The binding of the antagonist to the polymers resulted in a reduced acute toxicity.
