Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase

• Published in: Analytical chemistry (Washington) Vol. 73; no. 22; pp. 5415 - 5421
• Main Authors: Deng, Qing, German, Igor, Buchanan, Danielle, Kennedy, Robert T
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-11-2001
• Subjects: Adenosine - analogs & derivatives; Adenosine - chemistry; Adenosine - isolation & purification; Affinity Labels; Biological and medical sciences; Biotinylation; Chemistry; Chromatography, Affinity - methods; Chromatography, Affinity - standards; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Streptavidin
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0105437

A biotinylated-DNA aptamer (molecular weight 16 600) that binds adenosine and related compounds in solution was immobilized by reaction with streptavidin, which had been covalently attached to porous chromatographic supports. The aptamer medium was packed into fused-silica capillaries (50−150-μm i.d.) to form affinity chromatography columns. Frontal chromatography analysis indicated that the dissociation constants (K d) of cyclic-AMP, AMP, ATP, ADP, and adenosine were 138 ± 18, 58 ± 2, 38 ± 2, 28 ± 6 and 3 ± 1 μM, respectively, for aptamer immobilized on a controlled pore glass support. Similar values were obtained for aptamer immobilized on a polystyrene support except for a slightly higher K d for adenosine. The K d for adenosine is similar to the previously reported value of 6 ± 3 μM for adenosine−aptamer in solution indicating that immobilized aptamers can have affinity similar to that of the solution forms. Columns had 20 nmol of binding sites/100 μL of support media, which is 3.3-fold higher than that previously reported for immobilization of IgG on similar media, indicating that the aptamer can be immobilized with higher density than antibodies. Variation of mobile-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 complex mixtures such as tissue extracts.