Construction of 3′X55. The fluorescently labeled RNA constructs discussed in this paper were produced using a splinted-ligation method (Solomatin and Herschlag 2009). First, two RNA oligos were labeled with fluorophores and then annealed to a DNA splint before being ligated together via T4 RNA ligase II. The resulting product is an RNA–DNA hybrid containing 3′X55, where the donor fluorophore (Cy3B) is attached to nucleotide 13 and the acceptor fluorophore (CF660R) resides between nucleotides 39 and 40. Next, the DNA splint was digested with DNase I, allowing for 3′X55 to be purified using HPLC. Finally, the labeled RNA molecules were allowed to refold under storage conditions, where they appeared to form both 3′X55b (left) and 3′X55a (right). See Materials and Methods for more details.
