5'-CDPI3 MGB™ Phosphoramidite

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. CDPI₃MGβ-oligonucleotide conjugates have been found to be useful in the following applications:

• Arrest of primer extension and PCR blockers
• Short and fluorogenic PCR primers
• Real-time PCR probes
• miRNA Inhibitors

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.