Single Molecule FRET Measures Structure and Fast Dynamics of DNA and RNA Four-Way Junctions

Abstract

Which comes first, the deformation or the binding? High specificity of protein-DNA interaction is often related with specific deformation of the binding site. B-Z transition is the most dramatic structural change induced by protein-DNA interaction, where some segment of DNA abruptly changes from the right-handed B-DNA to the left-handed Z-DNA by the help of specific proteins. Here, we report single-molecule FRET studies on protein-induced Z-DNA formation. DNA duplexes with six CG-repeats were prepared. To monitor the conformational dynamics of the CG-repeat, we labeled Cy3 and Cy5 at each end of the CG-repeat. Surface-immobilized DNA molecules did not show any structural dynamics in normal physiological conditions. When a Z-DNA inducing protein, Za, was added to the buffer solution, however, fluorescence intensity increased abruptly without any accompanying FRET change. Abrupt FRET change occurred with time delay (~10 minute at 25 C on average). When the proteins were washed out, molecules didn't recover the original FRET value for more than 3 hours, but molecules without the FRET change readily recovered their original fluorescence intensity. From these result, we conclude that Za protein weakly interact with B-DNA, but the interaction becomes extremely strong once Z-DNA is formed. Next, we prepared a DNA duplex with methylated cytosine in the CG repeat. With millimolar Ni 2þ in the buffer solution, we observed the intrinsic B-Z transition dynamics, and Z-DNA stabilization by Za proteins. The transition time from B-DNA to Z-DNA, however, was not affected by the presence of Za proteins, which strongly support that Za protein induces Z-DNA by passively trapping Z-DNA structure transiently formed by the intrinsic B-Z transition dynamics. Even though we cannot directly observe Z-DNA, Z-DNA's are actually waiting there inside the cell to play their biological roles on time.

Single‐Molecule Metal‐Induced Energy Transfer (smMIET): Resolving Nanometer Distances at the Single‐Molecule Level

“…Subsequently, many studies have successfully used spFRET for studying biological problems on the single-molecule level; for example, see refs. [13][14][15][16][17][18][19][20][21].…”

Single-Molecule Metal-Induced Energy Transfer: From Basics to Applications

“…They used Near-field Scanning Optical Microscopy (NSOM) on immobilized short DNA fragments with attached FRET pairs. Subsequently, many studies have successfully used spFRET for studying biological problems at single-molecule level [40][41][42][43][44][45][46][47][48].…”