Biology Reference
In-Depth Information
Different materials applications have made use of the polarity of TMV
rods and the fact that the particles can be partially disassembled at one
end or the other. The helical encapsulation of the RNA molecule results in
sequence-definable 5
′
′
ends. Partial disassembly to expose the RNA
molecule at either end can be achieved. These methods were extensively
studied in the 1970s, and the reaction conditions to achieve partial
disassembly from 5
and 3
′→
′
′→
′
, respectively, of the viral rod are known
(Ohno & Okada, 1977; Wilson, 1978). To achieve stripping from the 5
3
and 3
5
′
end
mild sodium dodecyl sulfate (SDS) treatment can be used. Disassembly
from the 3
′
end is typically achieved using a mild dimethyl sulfoxide (DMSO)
treatment.
These methods have been exploited to specifically immobilize TMV
on solid supports. Spatially oriented assembly of partially disassembled
TMV with exposed RNA was achieved in a controlled manner. TMV was
disassembled selectively on the 5
′
end. The exposed RNA molecules were
then utilized for immobilization of the particles via nucleic acid hybridization
using complementary oligonucleotides bound on solid supports (Yi
,
2005). Furthermore, by using differentially labeled TMV particles and a
micropatterned substrate, the construction of a patterned TMV microarray
was achieved (Yi
et al.
, 2007). Immobilization and multilayer array
fabrication techniques are discussed in detail in Chapter 7.
et al.
3.6 GENERATION OF EMPTY AND NON-INFECTIOUS
PArtICleS
With developments in viral nanotechnology, there has been increasing
demand for empty particles or non-infectious particles. VLPs expressed in
heterologous expression systems are non-infectious as they lack genomic
nucleic acids; however, they typically pack random host nucleic acids.
Various
techniques have been developed that allow release and
removal of nucleic acids from capsids to generate particles that are truly
empty. These methods have also been adapted to packaging artificial cargos
into capsids (discussed in Chapter 5).
Empty particles, for example, can be self-assembled
in vitro
in vitro
from TMV or
CCMV coat proteins (Gillitzer
, 2007). Alternatively,
nucleic acid can be released by making use of the pH-dependent swelling
mechanism of particles, such as CCMV and HCRSV (Douglas & Young, 1998;
Ren
et al.
, 2006; Miller
et al.
, 2006). At pH 8.0, the particles undergo structural changes and
appear in a swollen and open conformation. The encapsidated nucleic acid,
which is RNA in both cases, can then be degraded by alkaline hydrolysis and
removed by differential ultracentrifugation. Mild denaturants, such as urea
or RNase (enzymatic degradation of the RNA) can also be added to ensure
degradation of the nucleic acids.
et al.
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