Combined triplex/duplex invasion of double-stranded DNA by "tail-clamp" peptide nucleic acid.
"Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension as determined by T(m) measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C(50) measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed that this was due to a dramatically reduced dissociation rate of such complexes. Increasing the PNA net charge also increased binding efficiency, but unexpectedly, this increase was much more pronounced for tailless-clamp PNAs than for tail-clamp PNAs. Finally, shortening the tail-clamp PNA triplex invasion moiety to five residues was feasible, but four bases were not sufficient to yield detectable dsDNA binding. The results validate the tail-clamp PNA concept and expand the applications of the P-loop technology.
Bentin
Thomas
T
Center for Biomolecular Recognition, IMBG, Department B, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark.
Larsen
H Jakob
HJ
Nielsen
Peter E
PE
D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D006860:Hydrogen Bonding; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D011742:Pyrimidine Nucleotides / Q000737:chemistry
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D020135:Peptide Nucleic Acids; D011742:Pyrimidine Nucleotides; C051802:triplex DNA; D004247:DNA