Aptamers are DNA (or RNA) oligonucleotides selected from random libraries of DNA sequences. They fold into a three dimensional structure to bind different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method selection.
We present in this note an alternative method of selection of aptamers using CE-UV-LEDIF detection/non-SELEX. Non-SELEX involves repeated rounds of partitioning using CE without the need for amplification and strand separation after each subsequent round.
Instruments | Capillary Electrophoresis: Agilent Technologies 7100 CE Detector: Picometrics ZETALIF LED 480nm/30nm |
Sample | T29 aptamers labelled with FAM (6-carboxyfluorescein) (fig 4) Thrombin protein is detected by UV absorbance (210nm) (fig 3) |
Labeling | DNA sample were denatured by heating at 95°C during 5min and let come to room temperature. Thrombin protein is then mixed with the T29 aptamers and injected into the capillary. All samples are diluted in a mixture of Tris-HCl 50mM + NaCl 100mM + MgCl2 1mM. |
Method | – Capillary: 90cm x 50 µm ID (effective length: 19cm) – Migration buffer: Na2HPO4 – Voltage: +20kV – Injection: 50mbars, 30s – Cassette temperature: 25℃ |
Double detection allows to determine the migration time of thrombin (TmUV=9 minutes, Fig3) and of T29 aptamer (TmLEDIF=18 minutes, Fig 4). The complex DNA target has an intermediate migration time (Fig 5).
Conclusion:
This application note shows the separation method of protein, complex DNA protein and DNA library. Double detection gives migration time of each component. The collection method by capillary electrophoresis is described in the application note AN 2.006.