Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg(2+) and Na(+) concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na(+)] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 +/- 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg(2+)] levels, which agrees quantitatively with the 32 +/- 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the K(D) value and Hill coefficient for [Mg(2+)]-dependent docking decrease significantly for [Na(+)] = 25 mM vs. 125 mM, indicating Mg(2+) and Na(+) synergy in the RNA folding process.
We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using biotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.
We report a new approach to an unidirectional photonic wire based on fluorescent dyes as chromophores and DNA as a rigid scaffold. The physical functioning of the wire is realized by dipole-dipole interaction, i.e. resonant energy transfer, between chromophores. The use of four dyes (Alexa 430, TAMRA, Cy3.5, and Cy5) with different excited state energies creates an energy cascade constituting the driving force of the energy current and providing the unidirectionality of the device. The unique molecular properties of DNA, its scaffold-like structure, combined with straightforward synthesis methods allowed the engineering of a 30 base pair double-stranded DNA with inter-dye distances of 10 base pairs (3.4 nm), respectively, a range where electronic interactions between the chromophores can be neglected but dipole-dipole induced fluorescence resonance energy transfer (FRET) is expected to be still highly efficient. Steady-state and time-resolved ensemble spectroscopic measurements show an overall energy transfer efficiency of ~ 0.60. That is, the unidirectional transport of photonic energy over a distance of ~ 10 nm and a spectral separation of ~ 250 nm. Furthermore, pulsed diode laser excitation at 440 nm in combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) was applied to characterize the effectiveness of individual photonic wires dispersed on glass coverslips.
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