CONFORMATIONAL STUDY OF BIOTINYLATED DNA OLIGONUCLEOTIDES UTILIZING 2-DIMENSIONAL NUCLEAR-MAGNETIC-RESONANCE AND MOLECULAR-DYNAMICS

The solution structure of DNA oligonucleotides containing a biotin gro up covalently attached through a linker arm to the 5' end has been stu died by two-dimensional NMR and molecular modeling, in an attempt to d etermine whether the biotin end group is accessible to avidin binding. Such experiments are useful in suggesting that purification of synthe tic DNA oligonucleotides by avidin-biotin affinity chromatography is c arried out with a minimum amount of intramolecular association of biot in with potential binding sites within the DNA. Two DNA oligonucleotid es that form hairpin structures have been proposed as possible worst c ase scenarios where biotin would not be available for avidin binding. These structures are d(XGCGCGTTTTCGCGC) and d(XGCGCGTTTTCGCGCAAAAA), w here X represents the biotin and linker structures. No NOEs were detec ted between the biotin/linker portion of the molecule and the DNA hair pin. 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 hai rpin, suggesting that the biotin/linker undergoes considerable additio nal motion compared to the DNA hairpin. Molecular dynamics calculation s also show that the biotin/linker undergoes a considerable range of m otion. Thus, all data indicate that the biotin is not immediately asso ciated with the hairpin and should be available for binding to avidin chromatographically relevant solvent conditions. 1995 Academic Press, Inc.