Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates.
DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.
Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, D-12489 Berlin, Germany.
Simon
S
Ficht
Amos
A
Mattes
Oliver
O
Seitz
D001483:Base Sequence; D002234:Carbodiimides / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D004252:DNA Mutational Analysis; D005998:Glycine / Q000737:chemistry; D006801:Humans; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013698:Templates, Genetic
D002234:Carbodiimides; D009841:Oligonucleotides; D010446:Peptide Fragments; D020135:Peptide Nucleic Acids; D010455:Peptides; D004247:DNA; D005998:Glycine