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Alkaline hydrolysis is also an effective method for particles that do not
undergo extensive swelling, such as CPMV. Alkaline hydrolysis can be applied
either to purified CPMV particles (Ochoa
et al.
, 2006) or during the initial
steps of the extraction protocol (Phelps
, 2007a). The reported shelf-
life of empty particles generated by alkaline hydrolysis is about 2 weeks
(Ochoa
et al.
, 2006), which limits potential long-term applications.
The nucleic acids of T7 phage can also be extracted by alkaline hydrolysis
(Liu
et al.
, 2006); nucleic acid extraction via osmotic shock has been applied
as an alternative method (Liu
et al.
, 2005).
Some viruses form empty particles during the natural infection process.
CPMV particles, for example, form empty as well as nucleic acid-containing
particles
et al.
. CPMV particles form three different components
that have identical protein composition but differ in their RNA contents
(Bancroft, 1962; Bruening & Agrawal, 1967; Wu & Bruening, 1971);
these components can be separated by density (Fig. 2.10). The particles
representing the top component are devoid of RNA, whereas the middle
and bottom components each contain a single RNA molecule, RNA-2 and
RNA-1, respectively (Lomonossoff & Johnson, 1991). However, the yield of
empty particles in a wild-type virus preparation is rather small (<10 %).
Empty, so-called ghost T7 phages can also be isolated (in low yields) at the
early stage of infection (Studier, 1972).
To address the need for non-infectious particles for safe use in materials
and medical applications, scientists have also developed methods to render
intact, genome-containing particles non-infectious. Short-wave (254 nm)
UV irradiation using a dosage of 2.0-2.5 J/cm
in planta
has been demonstrated as an
efficient strategy to crosslink the RNA genome within intact CPMV particles.
The particles were non-infectious for plants, remained intact, and maintained
chemical reactivity and cellular binding properties (Rae
2
, 2008). UV
inactivation of CPMV prior to or after modification thus provides a route for
designing non-infectious particles for safe use in
et al.
applications or as
materials. UV inactivation is a common technique and has been applied to a
wide range of viral pathogens (reviewed in Lytle & Sagripanti, 2005).
in vivo
. “SMArt” deSIgn oF MutAnt VnPs
An advantage of VNPs and other proteinaceous nanomaterials over any
synthetic material is that the biological material allows tuning using either
genetics or chemistry. Chemical modification strategies can also be applied
to synthetic materials, although high level of spatial control can typically
only be achieved using biomolecules. The genomes of viruses are small, and
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