Florescence Resonance Energy Transfer (FRET) describes a mechanism that energy may transfer between two closed chromophores through a non-radioactive dipole-dipole interaction. Once excited by a laser, the electrons absorb a photon (blue line in Figure 1.7) in the donor chromophore jump from the ground state (S0) to its higher vibrational
level of the first excited state (S1). Then the electrons relax energy rapidly to a lower vibrational level of S1 within picoseconds (ps), and gradual (nanoseconds) decay to the ground state in the form of fluorescence (green arrow in Figure 1.7). As an alternative, the energy of the donor excited state can be transferred nonradiatively to the acceptor with resonance energy transfer (dashed black arrows). Then, the electrons of the donor
molecule in turn are excited to their first excited states (S1), which can decay to the ground state and emits a photon in the form of fluorescence (red arrow in Figure 1.7). 105 As mentioned, the conditions for FRET to occur is the overlap between the donor emission and acceptor absorption spectra. For example, in our case, an argon gas laser with 514 nm is used, while the excitation range of Cy3 is from 500 nm to 550 nm (yellow area), which is perfect match. Meanwhile, there is one overlap (green area) between the emission of Cy3 and excitation of Cy5 (see Figure 1.8). The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET extremely sensitive to small distance changes between 1-10 nm range, leading FRET act perfectly as 'A spectroscopic ruler' to investigate molecular interactions. 106
(1)
The efficiency of FRET is expressed in Equation 1, where R0 is the Förster distance of
the two chromophores, at which, the FRET efficiency value is 0.5, indicating that half of the excitation energy is transferred to acceptor. R0 is a characteristic parameter of each
chromophores pair, can be calculated as the following equation (in Ängstrom).
(2)
where ØD is the fluorescence quantum yield of the donor in the absence of the acceptor, NAvo is Avogadro’s number, n is the refractive index of the medium, J(λ) is the overlap
integral describing the degree of overlap between the donor fluorescence emission
6 0 1 1 + = R R EFRET
spectrum and the acceptor absorption spectrum and ĸ 2 is the orientation factor, since the dipole moments of two chromophores rotate freely in all directions, in this case ĸ2 =2/3. In our sm-FRET measurements, we choose Cy3 and Cy5 as the two chromophores, Cy3 serves as the dye donor, while Cy5 serves as the dye acceptor. That's all due to Cy3 and Cy5 have relatively high photostability, high extinction coefficient and quantum yield, moreover, they have relatively small size, and thus easy to be conjugated to biomolecules. In addition, Cy3, which emits maximally at 580 nm and Cy5, which maximally excited at 640, the R0 of Cy3 and Cy5 is around ~6 nm. Indicating, if the
distance of Cy3 and Cy5 is around 5~6 nm, the efficiency value is around 0.5 which is a middle value, otherwise if the distance is larger than 2R0, like10 nm, the efficiency value
is almost 0.
Figure 1.7 Illustration of electron vibrational energy states that occur during FRET. (Figure
Figure 1.8 Excitation (absorption)(blue) and emission (fluorescence)(red) of Cy3 and Cy5, the spectral overlap integral is in green. (Figure adopted form http://www.biotek.com/resources/articles/fluorescence-resonance-energy-transfer.html)
Single-molecule Florescence Resonance Energy Transfer (SM-FRET) spectroscopy107 which was first performed on biological measurements in 1996,108 has been widely used for unraveling molecule structures, dynamics and chemical kinetics in highly heterogeneous and complex bio-systems.109-111 SM-FRET has been successfully applied in the investigation of biochemical structural dynamics of proteins, 112,113 nucleic acids,114,115 fusion from the membranes116,117, as well as protein-protein, nucleic acids- nucleic acids, protein-nucleic acids interaction.106,118 Furthermore, a lot of novel sample preparation methods and spectroscopic have been developed to promote the SM-FRET studies of the complex molecular biology problem.119,120 SM-FRET spectroscopy allow one to observe one molecule at a time, then demonstrate the trajectories of this molecule during measurement time, providing a powerful skill to resolve the subpopulations and heterogeneity that the ensemble measurement can't solve.
investigation of NC- induced NA structural remodeling. Both the NC-chaperoned NA annealing and NC-induced DNA bending have been observed to be complex, heterogeneous biomolecular processes that involve multiple intermediates along different pathways and are associated with complicated kinetics/dynamics over multiple time- scales. The unique ability of single-molecule spectroscopy to study one molecule at a time allows for unraveling of the tremendous heterogeneity of this type of biomolecular systems.84,121 A major source of the heterogeneity is the distribution of stoichiometries for the NC-NA nucleoprotein complexes, i.e. with different numbers of copies of NC, DNA and RNA molecules. An additional source of dynamic heterogeneity is the distribution of NA secondary structures, which may even include some mis-folded and long-lived intermediate states. Single-molecule spectroscopic measurements provide considerably richer information about the complexity and heterogeneity of biomolecular processes than the ensemble measurements performed on bulk samples. Of course there are also disadvantages to single-molecule spectroscopy, especially the necessity to label the protein and/or NA molecules with appropriate fluorescent dyes, which may introduce additional complication to the spectroscopic signals due to the photoblinking, photobleaching, and other photophysical processes. Such complication, however, can be minimized by choosing appropriate time windows/intervals for data collection and using oxygen scavenger reagents to suppress the photoblinking and to generate longer-lasting fluorescence signals.85 Single-molecule spectroscopy also provides unique opportunities of probing the kinetics of NC-chaperoned NA structural remodeling processes in aggregation-free environments. A severe complication in investigating NC-induced NA structural remodeling is the perturbations resulting from large scale NC/NA aggregation
(LSA).87,98,122-126 LSA refers to insoluble molecular aggregates composed of typically hundreds to thousands of NC and NA molecules. LSA can significantly alter the molecular structures, conformational dynamics, and stoichiometry associated with NC- NA interactions. LSA of this type may have been present in most of the in vitro ensemble measurements of NC-chaperoned processes. LSA has been described as both a biologically relevant effect that accelerates NA/NA annealing98,122,123 and, in contradiction, as an artifact of in vitro sample preparation that actually inhibits annealing.60,127,128 LSA occurs preferentially at high NC and NA concentrations (e.g. [NC] > 300 nM; [NA] > 50 nM) in vitro, however, the in vivo case differs dramatically in two major respects from the in vitro measurements. First, there is a low NA copy number
in vivo, two genomic RNA strands, not hundreds or thousands of separate NA as in LSA. Second, the RNA strands in vivo are of course much longer than the in vitro constructs. Recent single-molecule spectroscopic studies on TAR sequence annealing in the presence of high concentrations of NC and NA revealed that the LSA formation was accompanied by a large drop in NC’s ability to chaperone NA annealing, even inducing a “stalling effect”, for example no detectable annealing reaction, above certain NA and NC concentrations.127 We hypothesize that LSA has little to do with the in vivo processes and should be strictly eliminated from experimental protocols for studying NC-induced local melting, annealing, and binding. It has been recently shown that LSA is formed by a nucleation controlled precipitation-like process, rather than a simple Ostwald ripening.127 Thus, if nucleation of aggregate growth is avoided, aggregation-free NA annealing and bending studies can be accomplished.90 Measuring individual surface-immobilized molecules reacted with freshly mixed protein/NA solutions in a novel flow cell system at
relatively low NA concentrations provide a unique way to substantially suppress the formation of LSA during NC-chaperoned NA annealing and NC-induced NA bending. The proposed single molecule measurements will be carried out under aggregation free conditions to eliminate the complications due to LSA.
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