polymerase in the presence of 2 mM substrate 2 and the
natural-base NTPs (Fig. S7, ESIy). The gel-purified transcript
(3 mM) was then treated with either the 488-N3 or 594-N3
reagent (90 mM each), and the products were analyzed on a gel
(Fig. 2b). Only the tRNA transcripts obtained from the
Ds-containing DNA template with substrate 2 were labeled
with the fluorescent azide reagents. The modification with non-
fluorescent 3-azido-7-hydroxycoumarin10 yielded its fluorescent
product, which emitted at 480 nm upon 358 nm irradiation
(Fig. 2c).
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The site-specific modification of the tRNA molecules was
confirmed by using biotinylated products (Fig. 2d and e). The
modification of the tRNA transcript with the biotin azide
reagent was examined by a gel-shift assay, and about 90% of
the transcripts were shifted by binding streptavidin (Fig. 2d).
The 50-32P-labeled transcripts with or without the biotin
modification were digested under alkaline conditions, and half
of the digested products were treated with streptavidin-coated
magnetic beads to remove the biotinylated fragments. The
remaining fragments were then analyzed by gel electrophoresis
(Fig. 2e). The positions of each band on the ladder were
determined by comparison with the ladder yielded by G-specific
nuclease T1 digestion. On the lane without the streptavidin
treatment of the digested products obtained by biotin modifica-
tion, the fragment ladders larger than 33-mer were considerably
shifted (Fig. 2e, lane 5) as compared to the control ladder
(Fig. 2e, lane 2), and on the lane with the streptavidin treatment,
the ladders larger than 33-mer disappeared (Fig. 2e, lane 6).
These results indicated the site-specific biotin labeling at posi-
tion 33 of the tRNA transcript.
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In conclusion, we have developed a site-specific labeling
method for large RNA molecules, by combining the unnatural
base pair transcription system and the conventional click
reaction of Cu(I)-catalyzed azide-alkyne cycloaddition. The
modification efficiency is significantly improved, relative to
that using amino-linked unnatural bases3d in oligonucleotides
with N-hydroxysuccinimides. A variety of functional groups
including bulky groups can be site-specifically and efficiently
introduced into RNA molecules. This labeling method is
performed by a one-pot T7 transcription reaction involving
the Ds–Pa pair, the ethanol precipitation of transcripts, and
click reaction with functional azide reagents (Fig. S8, ESIy).
Thus, this labeling method could be used for a wide range of
applications with site-specifically labeled RNA molecules.
This work was supported by the Research Fellowships of
the Japan Society for the Promotion of Science for Young
Scientists (to T.I.). This work was also supported by Grants-
in-Aid for Scientific Research (KAKENHI 19201046 to I.H.
and 24710256 to M.K.) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10835–10837 10837