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104 Cell-Penetrating Peptides: Processes and Applications
5.4.2.1 Covalent Linkage of NLS to Condensing Agents
NLSs are also covalently linked to components of nonviral gene delivery systems. 86-88
For a single chimeric peptide, one of the most important criteria is sequence organization.
In chimeric proteins containing a DNA-binding domain and an NLS,
competition between these two moieties for DNA binding is prevented, and the
accessibility of the NLS enhanced, by using neutral or anionic NLSs, or a longer
NLS sequence with negatively charged residues upstream or downstream.
The presence of an SV40 NLS sequence linked to pLL increases transgene
expression. 33,34 The association of fusion peptide with an NLS was shown to promote
both rapid plasmid delivery into the nucleus and transgene expression. 50,51 A recent
study has demonstrated that condensation of DNA with pLL-NLS can be used to
enhance transfection of polyplex targeted to cells expressing the melanocyte stimulating
hormone (MSH) receptor. A chimeric Gal4-α MSH fusion protein was
associated with an optimized SV40 NLS. The NLS was used to compensate the
antagonistic impact of GAL4 binding to DNA and nuclear targeting and to promote
nuclear transfer of genes. 69-70
5.4.2.2 NLS Linkage Directly to DNA
The influence of the number and localization of the NLS on the nuclear import of
exogenous DNA are essential criteria participating in the controversy over the effect
of the NLS. Several studies have developed strategies to attach an NLS directly to
plasmid DNA by using either photoactivation 88 or cyclopropapyroloindole for conjugation.
86 Although the NLS–DNA conjugate was shown to associate with importin,
such approaches offer no control as to the number of NLSs attached or their location
within the DNA, two parameters essential for the success of this strategy. A critical
number of NLSs (between 50 and 100, depending on the method used) attached to
DNA are required for efficient transgene expression. 88,86 However, poor delivery of
plasmids into the nucleus was observed when a large number of NLSs was linked
to DNA. 86-89
Another approach is to control the number and site of attachment of the NLS
at specific points in the plasmid to avoid loss of transgene expression; the location
of the NLS is an important parameter. A method described by Zanta et al. proposes
to modify the ODN with NLS using maleimide chemistry followed by ligation of
this site to a luciferase expression cassette to produce a vector containing a singleterminal
NLS. 87 This approach was shown to improve gene expression following
transfection, independently of the transfection system used, and also lead to peak
expression after 12 h instead of 24 h in the absence of NLS. 87 Another method
developed by Ludtke et al., involves the use of SV40 NLS conjugated to streptavidin
bound to a biotinylated DNA fragment. The authors demonstrated that streptavidin–NLS
promoted active nuclear uptake of linear DNA. However, this method
seems to be limited to short DNA fragments. 89
One NLS molecule seems to be sufficient to promote nuclear delivery of DNA.
From a mechanistic point of view, the NLS docks the DNA onto the nuclear pore
and the DNA is then translocated through the nuclear pore. As the DNA enters the