Fluorescently labeling synthetic RNAs

This protocol covers the steps required to incorporate N-hydroxysuccinamide (NHS) functionalized fluorophores into synthetic RNAs containing a residue derivatized with a primary amine. This method has been widely used to label RNA oligonucleotides that are used directly, targeted to a complementary RNA using base pairing rules, or covalently ligated to a RNA of interest (Ha et al., 1999; Hodak et al., 2005; Baum and Silverman, 2007; Sattint et al., 2008; Akiyama and Stone, 2009; Solomatin and Herschlag, 2009). While this technique is quite general, the details of a particular experiment can vary, therefore, it is always important to keep in mind that other labeling strategies are available and should potentially be considered.