ABSTRACT
The reaction of ester hydrolysis catalysed by a DNA duplex in a sequence specific fashion has been developed, which is the fastest and most high yielding in comparison with the known reactions of this type.
Subject(s)
Copper/chemistry , DNA Primers , DNA/chemistry , Nucleic Acids/chemistry , Organometallic Compounds/chemistry , Peptides/chemistry , Base Sequence , Catalysis , Esters/chemical synthesis , Esters/chemistry , Hydrolysis , Molecular Sequence Data , Molecular Structure , Time FactorsABSTRACT
Two ligand-intercalator-peptide nucleic acid conjugates (L-NADI-PNAs) have been synthesized. Affinity of these conjugates to their complementary DNAs was found to be affected by Zn(2+). The magnitude of this effect could be controlled by a variation of the ligand. Upon binding Zn(2+) the L-NADI-PNAs form positively charged ZnL complexes, which interact with the negatively charged DNA backbone. This electrostatic interaction stabilizes PNA/DNA duplexes. It has been found that Zn(2+) dependent stabilization takes place only if the ZnL complex has a higher total positive charge than the ligand. Linear correlation has been observed between Zn(2+) induced stabilization of PNA/DNA duplexes and difference of charges of the ZnL complex and the ligand.
Subject(s)
DNA/chemistry , Peptide Nucleic Acids/chemistry , Zinc/chemistry , Mass Spectrometry , Molecular ProbesABSTRACT
A catalytic DNA-templated reaction of hydrolysis of an ester group in an N-modified peptide nucleic acid, which is activated by a Cu2+ complex-PNA, has been discovered and optimized. Both the ester-containing PNA and the metal complex PNA bind neighboring sites on a template DNA. This brings the reacting groups (the ester and the Cu2+ complex) in proximity to each other and accelerates the hydrolysis of the ester approximately 500 times in comparison with its hydrolysis in the absence of the template. The hydrolysis reaction provides >10(2)-fold kinetic discrimination between DNAs that are different from each other at a single nucleotide position. Natural enzyme T4 DNA ligase is slightly less selective. On the basis of this reaction a fully homogeneous and sensitive assay for sequence-specific DNA detection has been developed (10 fmol DNA). Identification of one of four DNAs (variation at one position) can be done in a single experiment. Since the Cu2+ ion is tightly bound in an associate containing the ester PNA, the metal complex PNA, and the template DNA, application of this method in buffers containing other Cu2+-binding ligands, e.g., PCR buffer and physiological buffer, is possible.
Subject(s)
DNA/analysis , Peptide Nucleic Acids/chemistry , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methods , Base Sequence , Catalysis , DNA/genetics , Hydrolysis , Nucleic Acid Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Selectivity of the cleavage of single stranded over hybridized forms of internally modified disulfide-peptide nucleic acids (PNA) has been optimized using a series of phosphines and thiols, which have different sizes and charges. For the most selective cleaver found (tris-(carboxyethyl)-phosphine), reactivity of single stranded PNA is 33 times higher than that of the PNA-DNA duplex. Selectivity of single stranded disulfide-PNA cleavage has been explained in terms of electrostatic interaction between the substrate and the cleaver.
