Tethered particle motion

Tethered particle motion (TPM) is a biophysical method that is used for studying various polymers such as DNA and their interaction with other entities such as proteins. Tethered particle motion (TPM) is a biophysical method that is used for studying various polymers such as DNA and their interaction with other entities such as proteins. The method allows observers to measure various physical properties on the substances, as well as to measure the properties of biochemical interactions with other substances such as proteins and enzymes.TPM is a single molecule experiment method. TPM was first introduced by Schafer, Gelles, Sheetz and Landick in 1991. In their research, they attached RNA polymerase to the surface, and gold beads were attached to one end of the DNA molecules. In the beginning, the RNA polymerase 'captures' the DNA near the gold bead. During the transcription, the DNA 'slides' on the RNA polymerase so the distance between the RNA polymerase and the gold bead (the tether length)is increased. Using an optical microscope the area that the bead moves in was detected. The transcription rate was extracted from data.Since then, a lot of TPM experiments have been done, and the method was improved in many ways such as beads types, biochemstry techniques, imaging (faster cameras, different microscopy methods etc.) data analysis and combination with other single-molecule techniques (e.g. optical or magnetical tweezers). One end of a polymer is attached to a small bead (tens to hundreds of nanometer), while the other end is attached to a surface.Both the polymer and the bead stay in an aqueous environment, so the bead moves in Brownian motion. Because of the tether, the motion is restricted. Using an optical microscope and CCD camera, one can track the bead position in a time series. Although the bead is usually smaller than the diffraction limit, so the image is a spot which is larger than the bead itself (point spread function), the center of the spot represents the projection on the X-Y plane of the end of the polymer (end-to-end vector). Analyzing the distribution of the bead position can tell us a lot of information about the polymer. In order that the motion would be polymer dominated, and not bead dominated, one should notice that the excursion number, NR, will be less than 1: where r {displaystyle r} is the bead radius, L {displaystyle L} is the contour length of the polymer and l p {displaystyle l_{p}} is the persistence length (50 nm in physiological conditions) of the polymer. (It is possible to work also when N R > 1 {displaystyle N_{R}>1} , but it should be treated carefully.) Metallic beads (usually gold) scatter light with high intensity, so one can use very small beads (~40 nm diameter), and still have a good picture. From the other hand, metallic beads are not the appropriate tool for optical tweezers experiments. Polystyrene beads scatter light weaker than metallic (in order to get the same intensity as getting from 40 nm gold bead, the polystyrene bead should be ~125 nm!), but it has the advantage that it can be combined with optical tweezers experiments. The major advantage of fluorospheres is that the excitation wavelength and the emission wavelength are not the same, so dichroic filter can be used to give a cleaner signal. The disadvantage of the fluorospheres is photobleaching.