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. 2011 Feb 4;39(10):4405–4418. doi: 10.1093/nar/gkq1339

Figure 1.

Figure 1.

Tat(44–61) accelerates annealing of two complementary RNAs but does not promote strand displacement. (A) Scheme of the FRET-based combined annealing and strand displacement (SD) assay according to Rajkowitsch and Schroeder (44). Two short complementary RNAs, labeled with a Cy3- (donor) and a Cy5-dye (acceptor), respectively, are annealed in a microplate (phase I). The close proximity of the two dyes results in a fluorescence resonance energy transfer (FRET) when the donor dye is excited. Both fluorescent emission signals are measured at 1 s intervals using a microplate reader, and the FRET index is calculated as the ratio of the FRET to the Cy3-signal. Strand displacement is detected in phase II of the assay which is started through the addition of an unlabeled competitor RNA and results in a decrease of the FRET signal. (B) The assay was conducted with 21R RNA in the absence or presence of 1 µM Tat peptide and at 30°C. E. coli StpA (1 µM) was used as a positive control. For clarity, the FRET index was normalized to 1 (phase I and II) and 0 (only phase I).