5. Dissolve the residue in a known volume of autoclaved water
and quantify the coupled RNA product by measuring the
absorbance. Refer reported literature for the molar extinction
coefficient of each cross-coupled product [29] (see Note 15).
6. Confirm the identity of coupled RNA products by mass spectroscopy (see Note 16). If the product is fluorescent, then
analyze its emission properties. For example, see Figs. 4 and 5
for the fluorescence profile of coupled RNA products, which
are labeled with a fluorogenic dye, environment-sensitive fluorescent nucleoside probe [31, 32], and fluorescence
microscopy-compatible NBD dye [33].
4 Notes
1. Thaw T7 RNA polymerase and RiboLock on an ice bath.
Centrifuge the enzymes for few seconds (3–4 s). Do not vortex
the enzyme solutions as they may denature.
Fig. 4 Top: Image showing the fluorogenic Suzuki coupling of IU-labeled
transcript 13 with boronic ester 7 and the reaction product 15. The samples
were irradiated using 365 nm light source. Bottom: Emission spectrum of
boronic ester 7 and coupled RNA product 15. CPS ¼ counts per second. Part
of this figure has been reproduced by permission of Nucleic Acids Research:
Oxford Journals [29]
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Manisha B. Walunj and Seergazhi G. Srivatsan