Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate

A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]incole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA, Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized, Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site, Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA, Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group, A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer, We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement, These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents.