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]indole-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.
Afonina
I
I
Epoch Pharmaceuticals, Bothell, WA 98201, USA.
Kutyavin
I
I
Lukhtanov
E
E
Meyer
R B
RB
Gamper
H
H
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D007211:Indoles / Q000737:chemistry; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics; D011758:Pyrroles / Q000737:chemistry
D017931:DNA Primers; D004277:DNA, Single-Stranded; D007211:Indoles; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D011758:Pyrroles