Fig. 6

DNA and RNA annealing activity of RPL7. a Emission spectra of Rh6G-cTAR-Dabcyl. (I) Emission spectra of 10 nM Rh6G-cTAR-Dabcyl (black line) with 10 nM RPL7 (dotted line) or with 100 nM dTAR + 100 nM RPL7 after completion of the annealing reaction (dashed line). Inset scheme of closed doubly labelled cTAR: red star corresponds to Rh6G and black circle of Dabcyl. (II) Emission spectra of 10 nM Rh6G-cTAR-Dabcyl (black line) with 10 nM Gag (dotted line) or with 100 nM dTAR + 100 nM Gag after completion of the annealing reaction (dashed line). Inset scheme of closed doubly labelled cTAR. (III) Emission spectra of 10 nM Rh6G-cTAR-Dabcyl (black line) in the presence of 10 nM of a mixture of 5 nM Gag and 5 nM RPL7 (dotted line) or with 100 nM dTAR + 100 nM of the mixture of 50 nM Gag and 50 nM RPL7 after completion of the annealing reaction (dashed line). Inset scheme of doubly labelled cTAR with an arrow showing a slight fraying of cTAR. b Real time monitoring of cTAR-dTAR annealing. Kinetic traces for the reaction of 10 nM doubly labeled cTAR with 100 nM non-labeled dTAR in the presence of 100 nM of RPL7 (red trace) or 100 nM of Gag (blue trace) or 100 nM of a mixture containing 50 nM of Gag and 50 nM of RPL7 (green trace). Each curve corresponds to a single assay but is representative of four independent measurements. All assays were fitted by a bi-exponential equation and the values of the kinetic rate constants were reported in Table 1. Inset describes the scheme of cTAR-dTAR annealing. Formation of the duplex increases the distance between Rh6G and Dabcyl and thus restores Rh6G emission. c Real time monitoring of cTAR-TAR annealing. Kinetic traces for the reaction of 10 nM doubly labeled cTAR with 1 µM of non-labeled TAR in the presence of 1 µM of RPL7 (red trace) or 1 µM of Gag (blue trace) or 1 µM of a mixture containing 500 nM of Gag and 500 nM of RPL7 (green trace). Each curve corresponds to a single assay but is representative of four independent measurements. All traces were fitted by a bi-exponential equation and values of the kinetic rate constants were reported in Table 2. Inset describes the scheme of cTAR-TAR annealing