2.8. S1 and D8 Aptamer Affinity Purification

RNA affinity purification utilizing S1 and D8 aptamers was performed as described in [33,34]. In vitro transcribed RNA (11 μg) was renatured in TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) by heating at 56 °C for 5 min, 37 °C for 10 min, and incubating at room temperature for 15 min. Streptavidin-agarose (SigmaAldrich,) was prepared by washing 10 times in lysis buffer (50 mM HEPES pH 7.4, 10 mM MgCl₂, 100 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), Complete protease inhibitors (Roche) and resuspended to 50% slurry in lysis buffer. Sephadex matrix was prepared by swelling 0.5 g Sephadex G-200 (Sigma-Aldrich) in 40 mL lysis buffer overnight at room temperature. The Sephadex was then washed 3 times with lysis buffer and resuspended to 50% slurry. RNA (1 μg) was subjected to electrophoresis on a 1% agarose gel in Tris/Borate/EDTA (TBE) buffer to confirm an intact, homogenous RNA population. The remaining 10 μg of RNA was combined with 100 μL prepared streptavidin beads or Sephadex G-200 and 500 μL of lysis buffer. Samples were allowed to rotate at 4 °C for 4 h then subjected to centrifugation at ~25 rcf for 1 min to separate matrix, and supernatant was removed. Matrices were washed 5 times with 500 μL of lysis buffer, with rotation at 4 °C for 10 min. The matrix slurry was loaded directly onto a 1% TBE agarose gel containing ethidium bromide and subjected to electrophoresis to visualize RNA associated with matrix. As a comparison, the supernatant from the initial matrix separation (i.e., the flow-through) was also subjected to electrophoresis on the agarose gel. Elution of aptamer-tagged RNA was also carried-out by transferring matrix material slurry, following wash steps, to Ultrafree-MC HV centrifugal filter units (0.45 μm pore size (Millipore)) with 10 mM biotin (Sigma-Aldrich) or 50 mg/mL dextran (average molecular weight 9000–11,000 Da (Sigma-Aldrich)) in lysis buffer. Filter units were subjected to rotation at 4 °C for 1.5 h then subjected to centrifugation at ~7500 rcf for 2.5 min.

Isolation of recombinant viral RNA from infected cells was carried-out as described above, but the streptavidin matrix was first blocked with 10 μg avidin from egg white (Sigma-Aldrich). To generate lysates from infected cells, two 150 mm plates of HeLa cell monolayers were infected with wild type PV1, D8+702PV1, or S1+702PV1 at an MOI of 20 following two washes with PBS. After 30 min adsorption, DMEM-8% NCS was added to cells which were then placed in 37 °C incubator for 4 h. Formaldehyde cross-linking was incorporated where indicated as described in [35]: infected cells were washed twice with PBS then cross-linked with 0.2%–1% formaldehyde in PBS for 10 min at room temperature with shaking. Formaldehyde was quenched with 0.25 M glycine for 5 min at room temperature. Infected cells or infected and cross-linked cells were collected by scraping and pelleted at ~1500 rcf for 5 min, followed by washing twice with ice cold PBS. Pellets were resuspended in lysis buffer containing 20 U/μL RNasin (Promega). Cells were lysed by three rounds of sonication for 10 s followed by incubation on ice for 2 min. Lysates were pre-cleared by centrifugation and supernatant was incorporated into the isolation procedure.