Screening of oligonucleotide pools. (A) TAR-R06 aptamer complexes were monitored as in Figure 2 except that a 5' extended TAR (rA-TAR) was immobilized on the beads through a biotinylated anchor oligonucleotide (biot-dT) (Figure 2C). Increasing concentrations of biot-dT are incubated in the presence of 40 nM rA-TAR and 10 nM dig-R06. The results are representative of three independent experiments carried out in triplicate (mean ± SD). Maximal AlphaScreen® signal is obtained for stoichiometric concentrations of biot-dT and rA-TAR. (B) Monitoring the evolution of SELEX pools to the HCV IRES domain II (biot-DII) (Figure 2C). Aptamer populations from 7 SELEX rounds (T1 to T7) as well as the starting oligonucleotide pool (T0) were processed for AlphaScreen® analysis as described in the Methods section. AlphaScreen® signals are reported for each population as the mean of three independent experiments ± SD carried out in duplicate. (C) Monitoring the evolution of SELEX pools to the HCV IRES domain II (biot-DII) (Figure 2C) by SPR. Biot-DII was immobilized on a streptavidin-coated sensor chip (SAD200 m, XanTec Bioanalytics). The populations were prepared at 500 nM in the SELEX buffer and were injected over the surface at 20 μl/min.