Journal: International Journal of Molecular Sciences
Article Title: 2′-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP)
Figure Lengend Snippet: Representative probing experiments using 5′-endlabeled aptamer A011 RNA and analyzed by ( A ) 20% or ( B ) 10% denaturing PAGE. Lanes 1 and 13, undigested RNA control (con.); lanes 2 and 14, limited alkaline hydrolysis; lanes 3, 12, 15 and 24, partial digestion with RNase T1 under denaturing (denat.) conditions; lanes 4, 5, 16 and 17, with two concentrations of RNase T1 under native conditions; lanes 6, 7, 18 and 19, with two concentrations of RNase V1; lanes 8, 9, 20 and 21, with two concentrations of nuclease P1; lane 10 and 22, with nuclease S1; lanes 11 and 23, Pb2+-induced hydrolysis. For experimental details, see Materials and Methods. Alkaline hydrolysis bands and the corresponding structure elements are indicated at the left and right margins, respectively, according to the numbering system presented in Figure 2 (A011, center). ( C , D ) Illustration of prominent cleavage sites in the context of the secondary structure of A011, derived from probing experiments such as those shown in panels A and B . Symbol sizes suggests the relative intensity of cleavage bands based on visual inspection.
Article Snippet: Enzymatic digestions were performed as follows: RNase T1 (Thermo Fisher Scientific, Waltham, MA, USA) either in 20 mM sodium citrat (pH 5.0), 7 mM urea, 1 mM EDTA and 1 unit RNase T1 for 30 min at 55 °C (denaturing conditions) or in binding buffer (see above) with 0.5 or 0.05 units for 20 min at room temperature (RT); 0.01 or 0.05 units RNase V1 (Thermo Fisher Scientific Pierce) in binding buffer for 20 min at RT; 1 unit RNase S1 (Thermo Fisher Scientific, Waltham, MA, USA) in binding buffer containing 4.5 mM ZnSO4 for 20 min at RT; 0.01 or 0.05 units nuclease P1 (Sigma-Aldrich, St. Louis, MO, USA) in binding buffer containing 0.4 mM ZnSO4 for 20 min at RT.
Techniques: Polyacrylamide Gel Electrophoresis, Derivative Assay