Many intra-cellular processes are attributed to DNA-protein or RNA-protein interactions. In order to study those, single molecule detection methods can be used. We developed a relatively simple system for three dimensional (3D) single molecule detection by combining few known methods.
In Tethered Particle Motion (TPM) (see also in Wikipedia) technique, one end of a bio-molecule (such as DNA or RNA) is linked to a surface, while the other end is linked to an optical marker (gold nanobead in our case). The sample is placed in an aqueous environment and it moves randomly (Brownian motion) in a constrained volume limited by the DNA length. By tracking the particle position, the DNA end-to-end vector distribution is found, and its properties, such as its rigidity, can be characterized.
In order to track the particle’s motion in the Z direction, Total Internal Reflection Microscopy (TIR) is used. In total internal reflection, a weak electromagnetic field (evanescent field) is produced. The intensity of the evanescent field decreases exponentially with the distance from the surface. Due to the dependence of the intensity on the distance from the surface, it is possible to extract the height of the bead (Z direction) from the intensity of the light that is scattered from the bead.
The distribution in the lateral (xy) plane is known to be Gaussian, and was measured many times before. On the other hand, the axial distribution was shown theoretically to be Rayleigh-like, due to the asymmetry of the bounded surface, and was never measured before. Few attempts that were done, showed symmetric distribution, probably due to the relatively large bead and short DNA (Jensenius et. al., PRL 1997, Blumberg et. al., Biophys. J. 2005), or due to the long scanning-time of confocal microscope (Lehner et.al., PRL 2006).
Quantitatively, we measured the expected Rayleigh-like distribution (see figure 1). Nevertheless, when comparing simulation results of short DNA strands (less than 3 micrometer) to the theoretical solution that is based on random walk, a discrepancy is found. We studied the source of this discrepancy, and found that it is related to tangent-tangent correlations of short DNA chains. For longer DNA strands, the solution converges to the analytical solution.
Using Boltzmann distribution, one can correlate the distribution of the bead position (which gives the end-to-end distribution of the DNA) to the potential landscape. Using this we found the 3D potential of a bead tethered by DNA (see figure 2).
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- M. Lindner, G. Nir, S. Medalion, H. R. C. Dietrich, Y. Rabin and Y. Garini, Force-free measurements of the conformations of DNA molecules tethered to a wall, Physical Review E 83, 011916 (2011).
- M. Lindner, G. Nir, H. R. C. Dietrich, I. T. Young, E. Tauber, I. Bronshtein, L. Altman and Y. Garini, Studies of single molecules at their natural form, Israel Journal of Chemistry 49, 283-291 (2009).