- Thermal cycler.
- Confocal microscope (e.g., Yokogawa Electric CV1000).
- LC-MS system (e.g., Shimadzu LCMS-2010EV).
- 2.2μm, 350 mm analytical columns (e.g., Shimadzu GLC
Shim-pack XR-ODS). - Freeze dryer (e.g., Tokyo Rikakikai FDU-1200).
3 Methods
3.1 Preparation of
the BHQ1-Immobilized
Resin
- Place 0.5 mL of EAH Sepharose™4B resin in a Poly-Prep
Chromatography Column and allow to drain by gravity. - Wash the resin thoroughly with at least three bed-volumes of
dioxane–H 2 O (3:1, v/v). - Add 1.0μmol BHQ1 carboxylic acid, 0.10 mmol triethyla-
mine, and 0.10 mmol DMT-MM in 0.5 mL of dioxane–H 2 O
(3:1, v/v) to the drained resin, and shake the suspension at
room temperature overnight (seeNote 3). - Drain the coupling solution from the resin, and add 0.50 mmol
acetic acid and 0.50 mmol DMT-MM freshly prepared in
0.5 mL of dioxane–H 2 O (3:1, v/v) to cap unreacted amino
groups on the resin. - After overnight incubation, drain the resin and wash thor-
oughly with dioxane–H 2 O (1:1, v/v) followed by metha-
nol–H 2 O (1:1, v/v). - Resuspend the resin with methanol–H 2 O (1:3, v/v) to make a
50% slurry. Store at 4C until use.
3.2 Preparation of
BHQ1-Cy3 Probe
Synthesis of the BHQ1-Cy3 probe is summarized in Scheme1.- Deprotect the Fmoc group of 1.5μmol Fmoc-β-Ala-Wang
Resin (seeNote 4) with 20% piperidine in DMF three times
10 min. - The resin is then washed with DMF three times 10 min.
- To conjugate the linker, 7.4 μmol Fmoc-amido-dPEG4™-
acid, 7.4μmolN,N^0 -diisopropylcarbodiimide, and 7.4μmol
HOBt in 20μL of DMF are added to the resin and incubated at
room temperature for 1 h (seeNote 5) to obtain Compound 1. - Wash the resin with DMF three times 10 min.
- To deprotect the Fmoc group, the resin is treated with 20%
piperidine in DMF three times 10 min (seeNote 5). - Wash the resin with DMF three times 10 min.
Live-Cell RNA Imaging with a Small Molecule 309