Organometallic DNA‐B12‐Conjugates as Potential Oligonucleotide Vectors ‐ Synthesis, Structural and Binding Studies with Human Cobalamin‐Transport Proteins
We report on the synthesis and structural characterization of Co‐(dN)25‐Cbl and Co‐(dN)39‐Cbl, organometallic DNA‐B12‐conjugates with single DNA‐strands consisting of 25 and 39 deoxy‐nucleotides, respectively, and on binding studies of these two DNA‐cobalamin (DNA‐Cbl) conjugates to the three homologous human Cbl transporting proteins transcobalamin (TC), intrinsic factor (IF) and haptocorrin (HC). This investigation tests the suitability of such DNA‐Cbls for the task of eventual in‐vivo oligonucleotide delivery. Binding of DNA‐Cbl to TC, IF and HC was investigated in competition with either a fluorescent Cbl‐derivative and Co‐(dN)25‐Cbl, or radiolabeled vitamin B12 (57Co‐CNCbl) and Co‐(dN)25‐Cbl or Co‐(dN)39‐Cbl. Binding of the new DNA‐Cbl conjugates was fast and tight with TC, but poorer with HC and IF, extending our similar original finding with the simpler DNA‐Cbl, Co‐(dN)18‐Cbl. The contrasting affinities of TC vs. IF and HC for the DNA‐Cbl conjugates are rationalized here by a stepwise mechanism of Cbl‐binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl‐binding proteins to the DNA‐Cbl, first bound to the respective beta‐domains. This transition is fast with TC, but slow with IF and HC, with which a weaker binding results. The invariably tight interaction of the DNA‐Cbl conjugates with TC makes the Cbl‐moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells
Elena Mutti, Miriam Hunger, Sergey Fedosov, Ebba Nexo, Bernhard Kräutler
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