Authors:Choi YS Transfered by BOC Sciences
This protocol is used to synthesize ImpA which is required to make the intermediate reaction product of RNA/DNA ligases. This is desirable when the target molecule has a 5'-phosphate because use of an activated linker oligo allows ligation without ATP and avoids self-circularization of the molecule of interest.
Reagent Supplier Product # CAS #
Adenosine 5 -monophosphoric acid (5 -AMP) MP Biomedical 210008001 18422-05-4
Dimethylformamide (DMF) D-4551 68-12-2
Triphenylphosphine T84409 603-35-0
2,2 -Dipyridyldisulfide 43791 2127-03-9
Imidazole Sigma I2399 288-32-4
Triethylamine Sigma T0886 121-44-8
Sodium perchlorate 410241 7601-89-0
Acetone Sigma 154598 67-64-1
Anhydrous ethyl ether 346136 60-29-7
Thin layer chromatography (TLC) cellulose plates with a 254-nm fluorescence indicator (cellulose-F TLC) 06011
(NH4)2SO4 A4418 7783-20-2 100% ethanol Gold Shield
MgCl2 Hexahydrate Enzyme Grade Fisher BP214-500 7791-18-6
glycerol Fisher BP229-1 56-81-5 HEPES VWR VWR1481-04 7365-45-9 acetylated bovine serum albumin B8894 9048-46-8 KOH
Dissolve 174 mg (0.5 mmol) of 5'-AMP in 15 ml DMF. Keep a 50-μl aliquot for TLC analysis.
Dissolve 262 mg (1 mmol) of triphenylphosphine, 220 mg (1 mmol) of 2,2'-dipyridyldisulfide, and 170 mg (2.5 mmol) of imidazole in 15 ml DMF and 0.9 ml (2.5 mmol) triethylamine. Keep a 50-μl aliquot for TLC analysis.
Add the AMP solution dropwise to a vigorously stirred triphenylphosphine containing solution. Cover beaker and stir for 1.5 hr at room temperature under fume hood.
Precipitate the ImpA by adding the reaction mixture dropwise into a vigorously stirred solution of 1.1 g (9 mmol) sodium perchlorate, 115 ml acetone, and 55 ml anhydrous ethyl ether.
Let the precipitate settle to the bottom of the beaker for 1 hr and decant ∼150 ml of the supernatant without perturbing the precipitate. A large glass pipet connected to a pipetting aid may also be used to aspirate off the supernatant.
Once the volume has been reduced to ∼20 ml, resuspend the precipitate with the residual supernatant and transfer the suspension to 30-ml Corex tubes. Transfer the residual precipitate by rinsing the beaker with small volumes (5 ml) of acetone. Collect the precipitate by centrifuging 10 min at 3000 × g (5000 rpm with a Sorvall SS34 rotor), room temperature.
Remove the supernatant and wash the pellet two times by resuspending it with 10 ml acetone and then centrifuge 5 min at 3000 × g, room temperature.
Resuspend the pellet in 10 ml anhydrous ethyl ether and centrifuge 20 min at 3000 × g, room temperature. Dry the pellet overnight in a vacuum oven at 40C.
Store the dried powder up to 3 weeks at -20C protected from humidity. The yield of ImpA is ∼80 mg. The molecular weight of ImpA is 396.28 g/mol.
Perform quality control of synthesized ImpA