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Example for double labeling of 3′-terminal 2′-O-(2-azidoethyl) modified RNA. (A) Labeling scheme for the preQ1 riboswitch RNA from Fusobacterium nucleatum.44 (B) HPLC profiles of crude reaction mixture after N-hydroxysuccinimide (NHS) ester based Cy3 conjugation (left) and subsequent strain-promoted alkyne azide conjugation (SPAAC) of Cy5 (middle), LC-ESI mass spectrum (right). For HPLC and LC-ESI mass specrometry conditions, see Figure 2 caption; for dye structures, see Figure S2.
