The tripeptide of dihydropyrroloindole-carboxylate (CDPI3) is a minor groove binding (MGB) moiety derived from the natural product CC-1065 with strong DNA binding properties. Synthetic oligonucleotides with covalently-attached CDPI3 have enhanced DNA affinity and have improved the hybridization properties of sequence-specific DNA probes. Short CDPI3-oligonucleotides hybridize with single-stranded DNA to give more stable DNA duplexes than unmodified ODNs of similar length. CDPI3 MGβ-oligonucleotide conjugates have been found to be useful in the following applications:
The simplest approach to MGB probe design is to use an MGB support, add a quencher molecule as the first addition and complete the synthesis with a 5'-fluorophore. Alternatively, a fluorophore support could be used with the 5' terminus containing a quencher molecule followed by a final MGB addition at the 5' terminus. Glen Research offers 5'-CDPI3 MGB™ Phosphoramidite and 3'-CDPI3 MGB™ CPG.
5'-CDPI3 MGB phosphoramidite was found to be hydrophobic enough that it required 10% THF in ACN to go completely into solution at a 0.1 M concentration and required a 3 minute coupling time. Deprotection can be carried out in EtOH/NH4OH 1:3 (v/v) 17 hr at 55°C and CDPI3 MGBis compatible with GlenPak™ purification.
With the CDPI3 MGB CPG, the optimal results are obtained if UltraMild monomers and Cap A are used during synthesis along with 0.5 M CSO oxidizer. However, the use of standard monomers with iodine oxidation followed by deprotection with EtOH/NH4OH 1:3 (v/v) for 17 hr at 55 °C will give acceptable results.