A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA

Kruse, Susanne; Zhong, Silin; Bodi, Zsuzsanna; Button, James; Alcocer, Marcos J. C.; Hayes, Christopher J.; Fray, Rupert

The RNA oligonucleotides were synthesized using a standard 0.2 mM scale protocol, but with a 15 min coupling time for each nucleotide addition step. The polymer-bound oligoribonucleotide was transferred from the synthesis column to a 1.5 ml microfuge tube and suspended in MeNH 2 solution (1 ml). The mixture was heated to 65 uC for 10 min, cooled to room temperature (water/icebath) and centrifuged for 1 min (10,000 g). The supernatant was separated from the CPG beads, the beads were washed with RNase free water (2 3 0.25 ml), all supernatants were combined and dried (2 h under nitrogen stream, then freeze dried). The oligoribonucleotide was resuspended in anhydrous NEt 3 N3HF/NEt 3 /NMP solution (250 ml of a solution of 1.5 ml NMP, 750 mlNEt 3 and 1.0 ml NEt 3 N3HF), heated to 65 uC for 1.5 h, cooled to room temperature and quenched with 3M NaOAc solution (25 ml). n-BuOH (1 ml) was added to the mixture, which was then thoroughly mixed, cooled to 270 uC for 1 -2 h to encourage further precipitation and centrifuged for 30 min (4 uC, 13 000 g). The supernatant was removed, the pellet washed with 70% EtOH (2 3 500 ml) and then dried in vacuo (30 min). The dry precipitate was dissolved in RNase free water (1 ml) and desalted using a Nap TM -10 column following the standard protocol. The resulting solution was freeze dried over night leaving the oligoribonucleotide as a white foam/powder. Samples for MALDI-mass spectrometry were prepared as follows 36 : Dowex TM ion-exchange beads were rigorously cleaned with dilute HCl, washed with water, then treated with dilute NH 3 and finally washed with water again to generate Dowex-NH 4 1 . Diammonium citrate (DAC) (100 mg) was dissolved in water (1 ml) and HPA (34.8 mg) was dissolved in 1:1 acetonitrile/ water (1 ml). The HPA solution was filtered through Dowex-NH 4 1 and the DAC solution (100 ml) was added to prepare the matrix stock. Prior to MALDI-MS acquisition, matrix stock (20 ml) was mixed with Dowex-NH 4 1 (5 ml) and each oligoribonucleotide sample (1 ml) was mixed with Dowex-NH 4 1 (19 ml). After 30 min the matrix, and after drying, the sample solution (0.5ml) were spotted onto the sample well and allowed to dry prior to confirmatory analysis by MALDI-MS.