Emerging Oligonucleotide Therapeutics - Discovery On …
To investigate the structures of stable DBE-mediated AGT-DNA cross-links, the investigators incubated AGT with DBE in the presence of a 15 base pair GC-rich double-stranded synthetic oligonucleotide. Heating the resulting cross-linked sample at 90oC for 30 minutes destroyed any labile adducts, leaving only stable cross-links for further analysis. Although treatment with trypsin did not fully digest the cross-linked AGT, it did reduce the protein to a 12 amino acid peptide, suitable for mass spectrometry (Figure 3). As expected, the results confirmed the presence of molecules containing both the peptide and the oligonucleotide joined by an ethylene bridge, but the data provided no information on the exact site of cross-link formation on the oligonucleotide. To address this problem, the investigators repeated the experiment using a double stranded oligonucleotide with the sequence T5G2T4. In this case, mass spectrometric analysis using collision-induced dissociation yielded an ion with an m/z of 1450, consistent with the structure of the peptide linked to guanine through an ethylene bridge (Figure 3). Similarly, use of an oligonucleotide with the sequence (AT)6 yielded, upon mass spectrometric analysis, an ion with an m/z of 1474.6, indicating a structure comprising the peptide, adenine, and an ethylene bridge (Figure 3). These results indicated that DBE could mediate stable cross-link formation between AGT and both the adenine and guanine bases of DNA.
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This article by Eugene Lukhtanov of ELITechGroup Molecular Diagnostics introduces the tripeptide of dihydropyrroloindole-carboxylate (CDPI3), which is a minor groove binding (MGB) moiety derived from the natural product CC-1065 and exhibits strong DNA binding properties. Synthetic oligonucleotides with covalently-attached CDPI3 moieties 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 oligonucleotides of similar length. The author describes the general properties of CDPI3 MGB-oligonucleotide conjugates in detail, along with some specific applications: