Retention and separation of adenosine and analogues by affinity chromatography with an aptamer stationary phase

A biotinylated-DNA aptamer (molecular weight 16 600) that binds adenosine a nd related compounds in solution was immobilized by reaction with streptavi din, which had been covalently attached to porous chromatographic supports. The aptamer medium was packed into fused-silica capillaries (50-150-mum i. d.) to form affinity chromatography columns. Frontal chromatography analysi s indicated that the dissociation constants (Kd) of cyclic-AMP, AMP, ATP, A DP, and adenosine were 138 +/- 18, 58 +/- 2, 38 +/- 2, 28 +/- 6 and 3 +/- 1 muM, respectively, for aptamer immobilized on a controlled pore glass supp ort. Similar values were obtained for aptamer immobilized on a polystyrene support except for a slightly higher Kd for adenosine. The Kd for adenosine is similar to the previously reported value of 6 +/- 3 muM for adenosine-a ptamer in solution indicating that immobilized aptamers can have affinity s imilar to that of the solution forms. Columns had 20 nmol of binding sites/ 100 muL of support media, which is 3.3-fold higher than that previously rep orted for immobilization of IgG on similar media, indicating that the aptam er can be immobilized with higher density than antibodies. Variation of mob ile-phase conditions revealed that ionic strength and Mg2+ level had strong effects on retention of analytes while pH and buffer composition had less of an effect. It was demonstrated that the column could selectively retain and separate cyclic-AMP, NAD(+), AMP, ADP, ATP, and adenosine, even in comp lex mixtures such as tissue extracts.