Fig. 5.
Efficiency of energy transfer for Cy3, Cy5-labeled hybrid RNA/DNA duplexes as a function of duplex length. The duplexes were constructed by using the same Cy5-labeled DNA strands as before, hybridized to complementary Cy3-labeled RNA strands. EFRET was measured for each duplex species, from an ensemble of molecules in the steady state (A) or from phospholipid vesicle-encapsulated single molecules (B). The EFRET values are plotted (filled circles) as a function of helix length. The estimated errors are shown on the single-molecule data. The black lines show simulation of the data based on the geometry of an A form helix with a periodicity of 12 bp per turn and a rise of 3 Å/bp step. A fraction of freely mobile fluorophore of 12% was assumed from the time-resolved fluorescence measurements of Cy3 attached to an RNA/DNA duplex (see text). A refractive index of n = 1.33 and quantum yield of 0.35 were used for the efficiency calculations. For both datasets, a half-width for the distribution of lateral motion of 42% gave the optimal agreement with the experimental data. The data and simulations for the DNA duplexes are shown for comparison by using open circles and gray lines. The phase shift between the two helical forms is very clear.