Rotaxanes of a-cyclodextrin (CyD) capped by 1-adamantaneacetic acid and a deoxyribonucleotide have been synthesized and purified using reverse-phase (RP) HPLC. The thermodynamic parameters of the dethreading process of deoxyribonucleotides through a-CyD were successfully determined by a simple and convenient kinetic study of the process using RP-HPLC.CyDs are cyclic oligosaccharides composed of five or more a-d-glucopiranosides, and are a very popular target in supramolecular chemistry for their unique host-guest inclusion capability. [1,2] DNA is another interesting subject of supramolecular chemistry because of its accurate complementary base-pairing and its regularly formed double-helical structure. [3,4] Conjugation of CyD-supramolecules with DNA oligomers should widen the scope of applications of molecular-based nanomechanical devices. [5][6][7][8][9][10] For example, an application of DNA/CyD-rotaxane conjugate for single-molecular DNA sequencing using AFM has been recently proposed. [10] A functional molecular abacus may also be possible when CyD-polyrotaxane is combined with DNA nanostructures. [11] Despite such attractive future applications, there are few reports on the actual synthesis of DNA/CyD hybrids, particularly DNA/CyD-rotaxane conjugates. Recently, we have successfully prepared and isolated a DNA/ a-CyD-rotaxane conjugate by capping a pseudorotaxane formed with a-CyD and an axle-bearing DNA oligomer with 1-adamantaneacetic acid. [12] Although the desired rotaxane structure was satisfactorily confirmed by mass analyses, the detailed structure, such as the configuration of a-CyD in the molecule, has remained unclear because of the highly complicated structure of the DNA portion and also of the low yield of the product. In this study, we have syn-