FIGURE 1.
Re-engineering the RNA aptamer Spinach to be a sequence-dependent molecular beacon. All sequence blocks are designated as numbered domains (* indicates complementary domains). (A) The 9-bp stem (highlighted in brown) of the folded, 80-nt minimized Spinach aptamer (24-2-min) was designated domain 6* at its 5′-end and domain 6.1 at its 3′-end. (B) The 5′-end was extended by adding an 8-nt domain 5* (highlighted in blue) while the 3′-end was extended by adding a duplicate of domain 6.1 (termed 6.2) and two 8-nt domains, designated 5 and 2 (highlighted in blue), respectively. Sequences derived from Saccharomyces cerevisiae tRNAtrp (highlighted in light purple) flanked the engineered Spinach. RNA folding should result in domains 5 and 6.2 being contiguously paired, trapping the aptamer in a nonfluorescent conformation in which domain 2 remains unpaired. (C,D) Domain 2 serves as a toehold for strand displacement by a trigger sequence containing domains 2*–5*–6* (highlighted in orange), leading to refolding of the aptamer into its active conformation. Associations between trigger and Spinach.ST are indicated with a double-sided arrow, while the conformational change that Spinach.ST undergoes is indicated by a double-tipped arrow. All nucleic acid structures were generated using NUPACK (Dirks and Pierce 2003, 2004; Dirks et al. 2007; Zadeh et al. 2011).