Selection procedure. Each round of selection included the following steps (see Fig. 2): binding of RNA to DM002 beads (through base-pairing); washing of beads to remove unbound RNA; elution of bound RNA with tobramycin; and regeneration of DM002 beads for the next round of selection. After each step, the beads were captured with a magnetic stand and the supernatant was removed. Binding, washing, and elution steps were all performed at 4 °C with pre-cooled solutions. Buffer for all steps was 50 mM Tris,
Tanespimycin 7.4, 500 mM NaCl (binding buffer). Hundred microliter of DM002 beads were washed twice with 200 μL of binding buffer and resuspended in 100 μL of binding buffer containing 30 μg of 32P-labeled RNA. Sample was heated at 65 °C for 5 min and cooled slowly to room temperature with occasional mixing. Sample was rotated overnight at 4 °C. Beads were quickly rinsed twice with 200 μL of binding buffer and washed extensively by rotating in 200 μL of binding buffer. Beads were washed 4 times for 15 min and once for 30 min. Bound RNA was eluted by rotating for 30 min in 200 μL of binding buffer containing tobramycin (see Table 1 for tobramycin concentrations). Beads were regenerated by suspending in 400 μL of binding buffer and heating at 65 °C for 5 min. A fresh aliquot of DM002 beads was used after 4 rounds of selection. Amount of RNA in each supernatant was determined by counting an aliquot in a scintillation counter. Amount of RNA
biomass bound specifically (through base-pairing) to the DM002 beads was calculated as the amount of RNA that eluted with tobramycin plus the amount of RNA that was removed by heating the beads at 65 °C.
