3. Use spin filters (0.45 μm) to filter the samples before injecting
on to the HPLC column.
4. Mobile phase A: 50 mM TEAA buffer, pH 7.0.
5. Mobile phase B: Acetonitrile. Filter the HPLC-grade acetonitrile through 0.22 μm filter paper.
3 Methods
3.1 Incorporation
of IUTP into RNA
Transcript by In Vitro
Transcription Reaction
(Fig. 3)
1. Thaw transcription buffer, reagents, and enzymes necessary for
transcription reaction on an ice bath.
2. Spin, vortex, and spin the buffer and reagents, and keep them
on ice bath (see Note 1).
3. Perform large-scale transcription reaction in 250 μL reaction
volume. Pipette 72.50 μL autoclaved water and 50 μL transcription buffer (5Â) into a 1.5 mL microcentrifuge tube.
4. Add 25 μL of 20 mM NTPs-UTP mix, 25 μL of 20 mM IUTP,
17.5 μL of 200 mM MgCl 2 , 2.5 μL of 1 M DTT, and 15 μL of
5 μM promoter-template DNA duplex.
Fig. 3 Incorporation of IUTP 1 by in vitro transcription reaction using T7 RNA polymerase, template DNA ON 12,
and T7 promoter 11. The sequence of IU-modified RNA transcript 13 is shown, which upon posttranscriptional
Suzuki coupling with boronic acid/ester substrates 4, 7, 9, and 10 gives coupled RNA ON products 14, 15, 16,
and 17, respectively. Part of this figure has been reproduced by permission of Nucleic Acids Research: Oxford
Journals [29]
Labeling RNA for Conformational Analysis and Imaging
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