6. ADHP (L1) can be purchased from a supplier. DMADHP
(L2) can be synthesized as described [34]. The catalyst is stable
when stored at À20 or À40
C. Precipitation was observed
when stored at RT for more than a month. We preferred to use
freshly prepared 10 mM solution from 50 mM stock solution
stored at À40
C.
7. We have observed deiodination of the IU-labeled transcripts if
the transcription reaction is performed for longer time. 6 h of
incubation time gave 10–12 nmol of the IU-labeled transcript
without detectable deiodination of the RNA transcript. So we
recommended shorter reaction time (e.g., 6 h) and wrapping
the microcentrifuge tube with aluminum foil.
8. We preferred not to add bromophenol blue (BPB) in the
loading buffer so as to avoid any adventitious contamination
of BPB in the transcript. To track the electrophoresis, we
loaded an extra lane with BPB in the same denaturing buffer.
9. After optimization, we found that this concentration and ratio
of IU-labeled RNA 13, boronic acid/ester substrate, and catalytic system gave the best results.
10. Depending on the boronic acid/ester substrate the reaction
time and yield could differ.
11. Filter the reaction mixture before injecting into the HPLC
column. Otherwise fine particles of Pd formed in the reaction
could affect the performance of the HPLC column.
12. We preferred HPLC purification of Suzuki-coupled RNA products as opposed to gel electrophoretic purification because it
was observed that Pd chelated to the RNA was not easily
removable by PAGE. However, with TEAA buffer system we
were able to successfully isolate the pure coupled products
by HPLC.
13. In our experiments, using the substrates listed in Fig. 2, we
used the conditions given in Subheading 3.4. These conditions
provide good separation between the coupled RNA product,
IU-labeled RNA substrate, deiodinated RNA, and boronic
acid/ester substrates. However, the HPLC purification
method could vary depending on the substrate and product,
and hence the readers are recommended to optimize the purification conditions.
14. Apart from monitoring the run at 260 nm (λ max of RNA), it
would be useful to monitor HPLC chromatogram at the characteristic absorption wavelength of the probe attached to
boronic acid/ester. A peak corresponding to the retention
time that absorbs at 260 nm and also at the characteristic
wavelength of the probe indicates that the fraction corresponds
to the coupled RNA product.
484
Manisha B. Walunj and Seergazhi G. Srivatsan
(L2) can be synthesized as described [34]. The catalyst is stable
when stored at À20 or À40
C. Precipitation was observed
when stored at RT for more than a month. We preferred to use
freshly prepared 10 mM solution from 50 mM stock solution
stored at À40
C.
7. We have observed deiodination of the IU-labeled transcripts if
the transcription reaction is performed for longer time. 6 h of
incubation time gave 10–12 nmol of the IU-labeled transcript
without detectable deiodination of the RNA transcript. So we
recommended shorter reaction time (e.g., 6 h) and wrapping
the microcentrifuge tube with aluminum foil.
8. We preferred not to add bromophenol blue (BPB) in the
loading buffer so as to avoid any adventitious contamination
of BPB in the transcript. To track the electrophoresis, we
loaded an extra lane with BPB in the same denaturing buffer.
9. After optimization, we found that this concentration and ratio
of IU-labeled RNA 13, boronic acid/ester substrate, and catalytic system gave the best results.
10. Depending on the boronic acid/ester substrate the reaction
time and yield could differ.
11. Filter the reaction mixture before injecting into the HPLC
column. Otherwise fine particles of Pd formed in the reaction
could affect the performance of the HPLC column.
12. We preferred HPLC purification of Suzuki-coupled RNA products as opposed to gel electrophoretic purification because it
was observed that Pd chelated to the RNA was not easily
removable by PAGE. However, with TEAA buffer system we
were able to successfully isolate the pure coupled products
by HPLC.
13. In our experiments, using the substrates listed in Fig. 2, we
used the conditions given in Subheading 3.4. These conditions
provide good separation between the coupled RNA product,
IU-labeled RNA substrate, deiodinated RNA, and boronic
acid/ester substrates. However, the HPLC purification
method could vary depending on the substrate and product,
and hence the readers are recommended to optimize the purification conditions.
14. Apart from monitoring the run at 260 nm (λ max of RNA), it
would be useful to monitor HPLC chromatogram at the characteristic absorption wavelength of the probe attached to
boronic acid/ester. A peak corresponding to the retention
time that absorbs at 260 nm and also at the characteristic
wavelength of the probe indicates that the fraction corresponds
to the coupled RNA product.
484
Manisha B. Walunj and Seergazhi G. Srivatsan