Biology Reference
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Wu
., 1995). The TR RNA stem loop is a regulatory element controlling
protein expression. When assembled MS2 VLPs, in solution or crystalline,
are soaked in a solution containing the TR operator, the operator diffuses
into the interior of the capsid through 1.4 nm-sized pores in viral capsid.
Within the capsid, one TR operator binds to each coat protein dimer; hence,
90 TR operators can be bound within an intact MS2 VLP (MS2 has
et al
T =
3
symmetry and consists of 180 identical copies of a coat protein).
The TR stem loop can be chemically modified, and studies have shown
that small therapeutic compounds such as the plant toxin ricin A chain or
5-fluorouridine can be covalently attached to the operator. Soaking VLPs in
a solution containing the drug-modified TR operators led to encapsulation
of the drugs within the VLPs.
studies using targeted drug-containing
MS2 VLPs confirmed specific delivery of the drug inherent with successful
cell killing of target cells (discussed in detail in Chapter 8) (Brown
In vitro
et al
.,
2002; Wu
et al
., 1995).
5.2 ENCAPSULATION OF MATERIALS DURING PARTICLE SELF-
ASSeMBly
Besides using the gating mechanisms of viruses, such as CCMV or RCNMV,
their well-understood
self-assembly mechanisms have also been
exploited for selective encapsulation of artificial nucleic acids or negatively
charged polymers; these techniques were pioneered by Bancroft
in vitro
et al
.
(1969).
self-assembly of VNPs is initiated by subtle electrostatic
interactions between the RNA and the protein subunits; the nucleic acid
assists and stabilizes particle formation. This basic principle can be exploited
by mixing coat protein monomers with negatively charged polymers and
dialysis against the appropriate assembly buffer. Capsid assembly is initiated
via electrostatic interactions of the coat proteins with the negatively charged
polymers and results in VLPs encapsulating the artificial cargo (Bancroft
In vivo
et
al
., 2007).
In the case of CCMV, assembled VLPs of differing sizes were obtained
by mixing the coat protein monomers with different-sized polymers.
Polystyrene sulfonate (PSS) of molecular weights ranging from 400 kDa to
3.4 MDa was mixed with CCMV coat proteins and VLPs were re-assembled.
Two distinct sizes of CCMV VLPs containing PSS polymers were formed:
22 nm-sized particles with
., 1969; Hu
et al
., 2008; Sikkema
et al
2 symmetry were obtained when
adding polymers with a molecular weight lower than 2 MDa; 27 nm-sized
VLPs resembling
pseudo T =
3 symmetry were formed when polymers of 2 MDa or
higher masses were mixed with the coat protein subunits (Fig. 5.2) (Hu
T =
et
al
., 2008).
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