Liposomes for DNA Nanotechnology: Preparation, Properties, and Applications - DNA Nanotechnology: From Structure to Function - page 62

cannot be accurately controlled. The free energy of a cholesterol molecule in
a lipid bilayer is estimated to be 23 k B T lower than that in water [ 25 , 26 ].
To improve the affinity, H o ok et al. developed a dual-cholesterol anchoring strategy
by hybridizing two single-cholesterol-labeled DNAs [ 27 ]. This is an example of
bivalent interaction being much stronger than single binding [ 28 ]. In this work,
a 15-mer DNA and 30-mer DNA with cholesterol groups at the 5 0 and 3 0 ends,
respectively, were hybridized to form 12 base pairs. Each DNA contains a three-
cytosine spacer between the cholesterol label and the duplex region. Using quartz
crystal microbalance with dissipation (QCM-D), they showed addition of the single-
cholesterol DNA to a phospholipid bilayer coated on a SiO 2 surface saturated at
100 nM, whereas addition of the dual cholesterol duplex saturated at 5 nM. In
addition, the liposome on the single-cholesterol DNA could be rinsed away, while
the dual-anchored DNA remained stable and appeared to be irreversible over the
duration of one hour. However, it is less straightforward to design such bivalent
probes, and the hybridization sequence is farther away from the liposome surface. In
addition, since DNA denatures at high temperature, stability might be compromised
under such conditions.

4.5.2 Cholesterol-PEG-Labeled DNA

In several reports, a polyethylene glycol (PEG) spacer was introduced between the
cholesterol label and the DNA. Sugaware et al. synthesized a cholesteryl-PEG-
labeled DNA, where the PEG molecular weight was 4,400 [ 22 ]. To achieve this, they
treated cholesteryl-PEG containing a succinic anhydride on the PEG end with N -
dimethylaminopyridine resulting in an active ester. The DNA component contained
a5 0 aminohexyl group, which can attack the active ester to form an amide bond. The
addition of this cholesteryl-PEG DNA in the presence of a lipid mixture to form
GUVs via electroformation resulted in its incorporation into giant vesicles. A more
popular form is the cholesteryl-tetraethylene glycol (TEG)-labeled oligonucleotides
since they are commercially available. In this case, the PEG chain is significantly
shorter with a molecular weight of only 240.
A cholesterol label can significantly condense the membrane lipid, while
cholesterol-TEG was reported to be free of this problem. Because of its commercial
availability, a number of systematic studies have been carried out using cholesteryl-
TEG-labeled DNA. Banchelli et al. reported that on each 70-nm POPC liposome, up
to five hundred 18-mer DNA with cholesterol-TEG label can be attached [ 29 ]. The
DNA conformation changes depending on the DNA density, where at low density,
DNA adopts a random coil conformation but becomes more rigid and extended
at higher DNA density. Beales and Vanderlick reported that the DNA localized
preferentially into the fluid phase of giant vesicles made up of 1:1 DLPC:DPPC or
DLPC:DMPE [ 30 ]. For another lipid formulation with DOPC/DPPC/cholesterol,
the DNA partition was less straightforward, where DNA stays in both phases.
Similar conclusions have also been observed by Bunge et al. [ 31 ].