Liposomes for DNA Nanotechnology: Preparation, Properties, and Applications - DNA Nanotechnology: From Structure to Function - page 63

4.5.3 Other Synthetic DNA/Lipid Conjugates

In addition to cholesterol-based modification, many other strategies have been
developed to hydrophobically modify DNA. For example, Boxer et al. synthesized
an amphiphilic oligonucleotide by reacting the 5 0 end of DNA with an iodination
reagent, (PhO) 3 PCH 3 I, making this end electrophilic to react with lipid-thiolate.
With subsequent deprotection, cleavage, and HPLC purification, the final product
was obtained [ 32 , 33 ]. Addition of this DNA (dissolved in a ratio of 1:1 buffer
and acetonitrile) to premade liposomes such as PC/DPPS at 4 ı C for 4 h resulted
in spontaneous and quantitative insertion into the liposome. Further purification
using gel filtration chromatography showed the presence of very little free DNA
in solution. In another example, Vogel et al. reported a cyclic scaffold, where two
long alkyl chains and the DNA are anchored [ 34 ]. However, these modifications are
not commercially available and require extensive organic synthesis and purification,
limiting their wide applications.

4.5.4 Reactive Lipids

An alternative method is to introduce an oligonucleotide to a vesicle via bioconju-
gation. In this case, a small percentage (e.g., 1-5%) of a reactive lipid is included
in the liposome formulation prior to the bioconjugation step. After formation of the
liposomes, DNA modified with a particular reactive group such as thiol or amino
is added, resulting in DNA attached only to the outer leaflet of the bilayer. For
example, Willner et al. reported the use of maleimide in lipid head group (MPB-PE)
for conjugation (Fig. 4.4 b) [ 35 ]. Liu et al. incorporated 5% of MPB-PE containing
lipid in the lipid mixture [ 36 ]. Once the liposomes are formed, it can be treated
with thiolated DNA. The advantage of this method is both the 3 0 and 5 0 thiol-
modified DNAs and the maleimide lipid are commercially available. Liu et al.
reported a DNA coupling efficiency of 25% [ 36 ]. Therefore, a significant amount
of free DNA needs to be removed. Boxer et al. used 1,2-dipalmitoyl- sn -glycero-3-
phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (sodium salt) (N-PDP-PE)
to react with a thiolated DNA to form a disulfide bond (Fig. 4.4 a), which is reversible
[ 37 ]. The disulfide exchange occurs in the presence of activated thiol DNA, which is
achieved by treating DNA with 10 M excess tris(2-carboxyethyl)phosphine (TCEP)
at pH 4. This activated thiol-DNA breaks the disulfide bond. Since the DNA
attached to the lipid is also by a disulfide bond, DNA can detach depending on
the medium pH. At alkaline pH, the disulfide functionality oxides to the sulfhydryl
group; however, at pH 7 the equilibrium lies more towards the disulfide.