This unit describes the specific incorporation of 15 N into the N7 and amino positions of adenosine (Basic Protocol 1), and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions (Basic Protocol 2). Two variations of the procedures are also presented that include either 12 C or 13 C at the C8 position of adenosine, and 13 C at either the C8 or C2 position of guanosine. These 13 C tags permit the incorporation of two 15 N-labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C-N coupling. While the major application of these specifically 15 N-labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. The Support Protocol describes the synthesis of 7-methylguanosine.
CAUTION:The procedures in this unit use a number of highly toxic and dangerous reagents. Raney nickel is pyrophoric when dry and may burst into flames if not kept wet. Phosphorous oxychloride (POCl 3 ) is very reactive and hydrolyzes to hydrochloric and phosphoric acids, which are both highly corrosive to skin and tissue. Cyanogen bromide is very dangerous; improper use of this reagent has led to deaths in the laboratory. All reactions must be done with great care in an appropriate chemical fume hood.