RNA for probing phosphates

Iodine scission of phosphorothioate containing RNA for probing phosphates 

The inertness of the phosphate groups to chemical probes under physiological conditions can be circumvented by co-transcriptional substitution of the phosphate with the more reactive phosphorothioate. The RNA is generated as described in Section 2.3.2 by in vitro transcription including 5% of the nucleoside 5 -0-(l-thiotriphos-phates) (NTPaS) in the reaction mixture. The subsequent iodine probing results in cleavage of the backbone that can be monitored by either end-labelling or reverse transcriptase analysis. 

Modification buffer 70 mM HEPES-KOH, pH 7.8 10 mM MgCl2 270 mM KC1 Iodine (2 mM, Sigma) 

Place 10 pmol renatured phosphorothioate-containing RNA or complex in 20 jxl modification buffer on ice. 

Terminate the reaction by adding 200 pi 0.3 M NaOAc (pH 6.0) and precipitate with 600 jjlI EtOH (leave on dry ice for 15 min). 

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