PNA-dependent gene chemistry: Stable coupling of peptides and oligonucleotides to plasmid DNA

Two approaches are described for stably conjugating peptides, proteins and oligonucleotides onto plasmid DNA. Both methods use a peptide nucleic acid (PNA) clamp, which binds irreversibly and specifically to a binding site cl oned into the plasmid. The first approach uses a biotin-conjugated PNA clam p that can be used to introduce functional biotin groups onto the plasmid t o which streptavidin can bind Atomic farce microscopy images of linearized plasmid show streptavidin localized at the predicted PNA binding site on th e DNA strand Peptides and oligonucleotides containing free thiol groups wer e conjugated to maleimide streptavidin, and these streptavidin conjugates w ere bound to the biotin-PNA-labeled plasmid. In this way, peptides and olig onucleotides could be brought into stable association with the plasmid. A s econd approach used a maleimide-conjugated PNA clamp. Methods are described for conjugating thiolated peptides and oligonucleotides directly to the ma leimide-PNA-DNA hybrid. This straightforward technology offers an easy appr oach to introduce functional groups onto plasmid DNA without disturbing its transcriptional activity.