Biomedical Engineering Reference
In-Depth Information
13.5.3 noncovalent delivery of strand displacement probes
by cationic nanoparticles
Another system based on quenched strand displacement probes was delivered by a
cationic shell cross-linked NP (cScK) (Fig. 13.11) [145]. This NP consisted of a
polystyrene core and a poly-N-2-aminoethylacrylamide shell, which is largely pro-
tonated at pH 7, though a number of amines remain unprotonated [146]. The cScK
served three purposes. The first was to electrostatically bind to the negatively charged
antisense turn-on probe. The second was to facilitate endocytosis through interaction
of the positively charged shell with the cell membrane. The third was to facilitate
endosomal destabilization by the pH sponge effect mediated by the remaining unpro-
tonated amines within the shell, thereby neutralizing protons during endosomal acid-
ification leading to an increase in osmotic pressure. The strand displacement probe
was designed to target iNOS mRNA, which is upregulated over 100-fold in response
to LPS/γ-IFN. The antisense binding site on the iNOS mRNA had been determined
through the use of an RT-ROL assay on endogenous mRNA recovered from whole
cell extracts of mouse macrophage cell line [71]. The strand displacement probe was
constructed from a neutral, high-affinity, degradation-resistant PNA strand bearing a
fluorescein fluorophore to a five-nucleotide shorter negatively charged partially com-
plementary DNA strand bearing a DABcYL fluorescence quencher. The remaining
five single-strand bases of the PNA strand serve as a “toehold” to facilitate binding
to the target mRNA sequence, which then causes displacement of the quencher strand
by branch migration. The higher affinity that the PNA strand has for the mRNA
strand due to the additional five base pairs that can form drives the exchange reaction
Intracellular
Extracellular
Endosome
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cSCK
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DNA
PNA
mRNA
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fIgure 13.11
cationic shell cross-linked nanoparticle-mediated delivery of a quenched
strand displacement probe system. In this system, a cationic nanoparticle electrostatically
binds to a quenched probe consisting of a neutral PNA hybridized to a partially complemen-
tary oligodeoxynucleotide. The NP then mediates entry of the probe into the cell via endocy-
tosis facilitated by its positive shell and subsequent escape from the endosome via the proton
sponge effect.
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