High Purification of tRNA Molecules After Affinity Chromatography and 3′ pCp-Cy3 Labeling

To obtain highly purified Alanine tRNA (UGC) molecules, we used the Streptavidin Sepharose^TM High Performance beads (GE Healthcare, 17-5113-01) coupled to the 3′-biotinylated tDNA Ala (TGC)-3′ probe (probe Ala TGC N° 3, as described).

We first mixed 10–20 μg of total tRNA with 1 nmole of tDNA probe and 40 U of RNaseOUT in 500 μl of annealing buffer [5X SSC (saline-sodium citrate); 0.05% SDS (sodium dodecyl sulfate)]. The mix was then incubated sequentially, 5 min at 70°C, 30 s at 4°C and 2.5 h at 42°C. Twenty μl of Streptavidin Sepharose beads were deposited in a 20 μm receiver column (Macherey-Nagel, 740522) and equilibrated with 3 × 500 μl of annealing buffer. Columns were incubated 15 min at room temperature in gentle shaking and 15 min at 42°C. They were centrifuged 15 s at 11000 g and then washed as follows: 2 × 500 μl of 5x SSC (5 min at 42°C), 1 × 500 μl 2x SSC (5 min at 42°C), 1 × 500 μl 2x SSC (15 min at 47°C), 1 × 500 μl 2x SSC (15 min at 52°C). After each washing step, the column was centrifuged 15 sec at 11000 g. tRNA elution was by adding 2 × 300 μl of elution buffer (10 mM Tris–HCl pH 7.5; 1 mM EDTA; 5 M Urea). Total elution was ethanol precipitated and resuspended in 2.96 μl of water for 3′ pCp-Cy3 (Jena Bioscience NU-1706-CY3) labeling.

Before labeling, we added 26% (v/v) DMSO (1,04 μl) to the purified tRNA (2,96 μl) and incubated 10 s at 100°C and immediately cooled on ice. The labeling reaction was performed using the Biolabs T4 RNA ligase (M0204). Ligase buffer and ATP were added to the tRNA-DMSO mix and ligation was performed with 20 μM of pCp-Cy3 and 5 units of T4 RNA ligase in 10 μl at 16°C overnight. The labeling was done in the same way for total tRNAs fraction, using 2.5 μg total tRNAs.

The different fractions were analyzed by electrophoresis on 7 M Urea-15% acrylamide gel and scanned on a GE Healthcare Ettan DIGE imager system. The bands corresponding to the purified tRNA were cut from the gel and eluted overnight at room temperature in 0.5 M ammonium acetate, 10 mM magnesium acetate, 0.1 mM EDTA and 0.1% SDS. After phenol extraction, tRNAs were ethanol precipitated and finally recovered in 8 μl of water.