Changes of weight-average molecular weights
(M
w) and the molecular weight
distributions
in terms of weight fraction (g
w) were evaluated
in the course of sonication for three nucleic acids in
different
conformations by the gel permeation chromatography/low angle laser
light scattering (GPC/LALLS)
method. High M
w samples used were
single-stranded poly(uridylic acid), (U)
n
,
double-stranded DNA,
and triple-stranded poly(adenylic acid)·2poly(inosinic
acid), (A)
n
·2(I)
n
,
in 0.2−0.1 M NaCl at 0 °C. The
experimental g
w vs M curves were
computer-simulated for different chain-scission mechanisms:
(1)
random, (2) midpoint, (3) Gaussian, (4) classical partially random, and
(5) newly proposed partially random
scission models. In comparison with the GPC/LALLS data, the middle
portion of each single- or multiple-stranded chain was sheared randomly, whereas the adjacent end portions
of the chain resisted sonic
scission. The M
w value of each intact end
portion was 1.0 × 105 for (U)
n
,
1.5 × 105 for DNA, and 1.7 ×
105 for
(A)
n
·2(I)
n
,
corresponding to the lowest possible M
w for the
sonicated but unfractionated nucleic
acid samples under the present conditions.
The nano-kinetic movement of a single DNA molecule was observed and analyzed by a newly developed video-microscope system with an optical fiber, called a pin-fiber video scope. A single lambda-DNA molecule was put in focus using fiber-illumination, and the stretching and shrinking motion was measured. The molecule's kinetics were analyzed by numerical calculations and are discussed. A photocleavage phenomenon of DNA molecules was also visualized by the pin-fiber video scope. The new video-microscope system has the potential to observe and analyze the nano-kinetics of a single molecule.
Particles of HPLC resins are used for the trapping of secreted molecules from a single cell. The basic molecules, e.g., histamine, are secreted from a single RBL-2H3 cell by granule exocytosis and are trapped by cation-exchange HPLC resins outside the cell. Since quinacrine is concentrated into the exocytotic and acidic microgranules in RBL-2H3 cells, which are used as a model cell line of mast cells, we measured the change in the fluorescence intensity of the quinacrine released from the cells and that of molecules trapped on the resin using a videomicroscope. By measuring the increase in the fluorescence intensity of the resins, we can estimate the real time course of molecular secretion from a single cell.
The single fiber-illumination method was applicable to visualize nanoparticle movement clearly and to estimate their sizes in solution. This simple method is suitable for the in situ observation of the nanoparticle-binding process to target cells.
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