Templated metal catalysis for single nucleotide specific DNA sequence detection.
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.
Boll
Iris
I
Anorganisch-Chemisches Institut, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany.
Krämer
Roland
R
Brunner
Jens
J
Mokhir
Andriy
A
D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000032:analysis* / Q000235:genetics; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020135:Peptide Nucleic Acids; D004247:DNA