16. Load the filtrate from step 14 onto the conditioned reversedphase Sep-Pak column.
17. Wash the column with 12 mL of autoclaved water and then
elute the transcript with 4 mL of 40% (v/v) acetonitrile in
autoclaved water. Collect ~1 mL fractions in 1.5 mL microcentrifuge tubes.
18. Record the absorbance of the fractions at 260 nm to confirm
the presence of the transcript.
19. Evaporate the fractions containing the transcript to dryness.
Dissolve the residue in a known volume of autoclaved water
and determine the concentration using the molar extinction
coefficient at 260 nm (ε 260 ¼ 84,300/M/cm) [29].
20. Confirm the purity and identity of IU-labeled RNA transcript
by HPLC and mass analysis. For details refer supporting information of reported literature [29].
3.3 Posttranscriptional
Suzuki-Miyaura
Cross-Coupling (Fig. 3)
1. Pipette 10 μL of 5Â Tris–HCl buffer and 7.5 μL DMSO into a
0.5 mL microcentrifuge tube. Add IU-labeled RNA ON 13
(9.1 μL of 550 μM stock, 5 nmol, 1 mol equivalent).
2. To the above reaction mixture add boronic acid/ester (2.5 μL
of 100 mM stock in DMSO, 50 equiv.). Add 19.9 μL of
autoclaved water.
3. Initiate the reaction by adding Pd(OAc) 2 (L1) 2 or Pd
(OAc) 2 (L2) 2 catalyst (1 μL of 10 mM stock, 2 equiv.). The
final reaction volume is 50 μL. The final concentration of RNA
13, boronic acid/ester substrate, catalyst, and DMSO% is
100 μM, 5 mM, 200 μM, and 20%, respectively (see Note 9).
4. Incubate the reaction mixture at 37
C for 6 h (see Note 10).
5. Quench the reaction by freezing the sample at À40
C.
3.4 HPLC Purification
of Functionalized RNA
Products
1. Filter the reaction mixture and wash the spin filter with 40 μL
of autoclaved water (see Note 11).
2. Analyze the filtrate by RP-HPLC (see Note 12).
Separate the cross-coupled product from the reaction mixture using following mobile phases and gradient: mobile phase
A: 50 mM TEAA buffer (pH 7.0); mobile phase B: acetonitrile.
Flow rate of 1 mL/min gradient: 0–30% B (100–70% A) in
35 min, 30–100% B (70–0% A) in 10 min, 100% B for 5 min,
and 100% A for 5 min (see Note 13).
3. Monitor the HPLC run at 260 nm and at the respective wavelength of each fluorophore (see Note 14).
4. Collect the fraction corresponding to the coupled RNA product (see Note 14) and lyophilize the buffer. Add water to the
residue and lyophilize again. This step removes the volatile
TEAA buffer efficiently.
Labeling RNA for Conformational Analysis and Imaging
481
17. Wash the column with 12 mL of autoclaved water and then
elute the transcript with 4 mL of 40% (v/v) acetonitrile in
autoclaved water. Collect ~1 mL fractions in 1.5 mL microcentrifuge tubes.
18. Record the absorbance of the fractions at 260 nm to confirm
the presence of the transcript.
19. Evaporate the fractions containing the transcript to dryness.
Dissolve the residue in a known volume of autoclaved water
and determine the concentration using the molar extinction
coefficient at 260 nm (ε 260 ¼ 84,300/M/cm) [29].
20. Confirm the purity and identity of IU-labeled RNA transcript
by HPLC and mass analysis. For details refer supporting information of reported literature [29].
3.3 Posttranscriptional
Suzuki-Miyaura
Cross-Coupling (Fig. 3)
1. Pipette 10 μL of 5Â Tris–HCl buffer and 7.5 μL DMSO into a
0.5 mL microcentrifuge tube. Add IU-labeled RNA ON 13
(9.1 μL of 550 μM stock, 5 nmol, 1 mol equivalent).
2. To the above reaction mixture add boronic acid/ester (2.5 μL
of 100 mM stock in DMSO, 50 equiv.). Add 19.9 μL of
autoclaved water.
3. Initiate the reaction by adding Pd(OAc) 2 (L1) 2 or Pd
(OAc) 2 (L2) 2 catalyst (1 μL of 10 mM stock, 2 equiv.). The
final reaction volume is 50 μL. The final concentration of RNA
13, boronic acid/ester substrate, catalyst, and DMSO% is
100 μM, 5 mM, 200 μM, and 20%, respectively (see Note 9).
4. Incubate the reaction mixture at 37
C for 6 h (see Note 10).
5. Quench the reaction by freezing the sample at À40
C.
3.4 HPLC Purification
of Functionalized RNA
Products
1. Filter the reaction mixture and wash the spin filter with 40 μL
of autoclaved water (see Note 11).
2. Analyze the filtrate by RP-HPLC (see Note 12).
Separate the cross-coupled product from the reaction mixture using following mobile phases and gradient: mobile phase
A: 50 mM TEAA buffer (pH 7.0); mobile phase B: acetonitrile.
Flow rate of 1 mL/min gradient: 0–30% B (100–70% A) in
35 min, 30–100% B (70–0% A) in 10 min, 100% B for 5 min,
and 100% A for 5 min (see Note 13).
3. Monitor the HPLC run at 260 nm and at the respective wavelength of each fluorophore (see Note 14).
4. Collect the fraction corresponding to the coupled RNA product (see Note 14) and lyophilize the buffer. Add water to the
residue and lyophilize again. This step removes the volatile
TEAA buffer efficiently.
Labeling RNA for Conformational Analysis and Imaging
481