Page 510 - Book Hosokawa Nanoparticle Technology Handbook

APPLICATIONS 16 NANOPARTICLE FORMATION OF DNA (GLOBULE TRANSFORMATION)

120 reagents [2]. Fig. 16.3 shows sequential photographs
of the globule transition in 15% PEG20000 solution
PEG concentration (mg/ml) 80 (B) (C) minum ion is generated at an aluminum anode, and
by aluminum ion supplied by an electrode reaction.
100
When DC voltage is applied to electrodes, the alu-
migrates toward the cathode (left direction in the fig-
ures) by electrophoresis. When the aluminum front
60
(dotted lines in Fig. 16.3) migrates across an area of
DNA molecules, globule transition is induced imme-
40
diately due to exceeding of a critical concentration of
the aluminum ion because the solution contains PEG.
(A)
20
As the result of the globule transition, blurred spots of
coiled DNA change to condensed blight spots as
0 shown in Fig. 16.3.
0 20 40 60 80 100 120 140
MgCl concentration (mM)
2
1. Tolerance of DNA nanoparticles against
mechanical stress
Since the globule structure is highly condensed, effect
of mechanical stress can be suppressed, and this prop-
coil and erty permits handling of giant DNA in solution as
coil globule globule
(A) (B) (C) demonstrated previously [3]. In this experiment, first
of all, yeast chromosomal DNA embedded in agarose
gel plugs was prepared. Some plugs were treated with
condensing reagents of combination of PEG and
Figure 16.1
Dependence of DNA structure on the concentration of NaCl for globule transition. After this treatment, the
PEG and MgCl . In PEG/MgCl , the state of the DNA is samples were stained with a fluorescent dye and
2
2
represented by: (A) coiled; (B) coiled/globule coexistence; observed with a fluorescent microscope. To evaluate
(C) globule. the effect of mechanical stress, the gel plugs on cov-
erslips were melted by increasing the temperature and
then immediately solidified by cooling. Effects of the
solution flow on the shape of DNA molecules were
evaluated because melting of agarose plug generates
shear stress upon DNA molecules accompanying the
flow in solution. Fig. 16.4 shows the shape of the
DNA molecules treated as described previously.
Fig. 16.4 demonstrates that globule transition con-
densed DNA molecules significantly, and the con-
densed structure remained even after exposure of
mechanical stress. On the other hand, coiled DNA
molecules were stretched by flow. This strongly sug-
gests that breakdown due to shear stress of flow can
be suppressed by globule transition.
This property was also examined by pulsed field
gel electrophoresis. Agarose gel plugs containing
coiled DNA and globule DNA were melted and
exposed to shear stress by vortexing solution at a
different speed. Those samples were re-solidified
and reverted to coiled structure by soaking gel plugs
in electrophoresis buffer, and analyzed by pulsed
field gel electrophoresis. As shown in the elec-
trophoregram of Fig. 16.5, solution mixing produced
many short fragments and DNA remained after mix-
ing in the case of coiled DNA no longer. On the
Figure 16.2 other hand, long DNA still remained after mixing in
Image of globule DNA captured by a transmission electron the case of globule DNA. This result suggests that
microscope (provided by Dr. Y. M. Urano, Toyohashi Univ. fragmentation of long DNA can be strongly sup-
of Tech.). pressed by globule transition, and globule transition

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