Journal: Nucleic Acids Research
Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7
Figure Lengend Snippet: Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.
Article Snippet: DNase treatment and purification of RNA fragments To obtain the purified, dephosphorylated native fragment, the DNA/RNA hybrid solution was incubated with 0.1 U/μl FastAP (Thermo Scientific) and 1.25 U/μl DNaseI (Thermo Scientific) for 1 h at 37°C.
Techniques: Generated, Polyacrylamide Gel Electrophoresis, Migration, Electrophoretic Mobility Shift Assay, Isolation, Fluorescence, Marker, Ligation