PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

Figure 4

T7 transcription for site-specific biotin labeling of 260-mer transcripts. Transcripts from each 282-bp DNA template containing 3′-TDsC-5′, 3′-GDsC-5′, 3′-ADsC-5′, or 3′-CDsC-5′, in the presence of the natural NTPs (2 mM) and Biotin-PaTP (2 mM), were analyzed on a 5% denaturing polyacrylamide gel (upper gel image). After the purification of each 260-mer transcript, gel-mobility shift assays were performed using streptavidin. Biotinylated RNA-streptavidin complexes were separated from free RNAs on an 8% nondenaturing polyacrylamide gel, and the amounts of the complexes (yields) were determined from the band intensities. The experiments were independently repeated twice, and representative data are shown. The gel-mobility shift values were averaged from two data sets.