Conformational study of biotinylated DNA oligonucleotides utilizing two-dimensional nuclear magnetic resonance and...

The solution structure of DNA oligonucleotides containing a biotin group covalently attached through a linker arm to the 5' end has been studied by two-dimensional NMR and molecular modeling, in an attempt to determine whether the biotin end group is accessible to avidin binding. Such experiments are useful in suggesting that purification of synthetic DNA oligonucleotides by avidin-biotin affinity chromatography is carried out with a minimum amount of intramolecular association of biotin with potential binding sites within the DNA. Two DNA oligonucleotides that form hairpin structures have been proposed as possible worst case scenarios where biotin would not be available for avidin binding. These structures are d(XGCGCGTTTTCGCGC) and d(XGCGCGTTTTCGCGCAAAAA), where X represents the biotin and linker structures. No NOEs were detected between the biotin/linker portion of the molecule and the DNA hairpin. In addition, the magnitude of the NOEs between neighboring groups in the biotin/linker was approximately an order of magnitude smaller than that of the neighbors at a comparable distance within the DNA hairpin, suggesting that the biotin/linker undergoes considerable additional motion compared to the DNA hairpin. Molecular dynamics calculations also show that the biotin/linker undergoes a considerable range of motion. Thus, all data indicate that the biotin is not immediately associated with the hairpin and should be available for binding to avidin under chromatographically relevant solvent conditions.