Synthesis of oligonucleotide analogues for use in DNA nanostructures

Abstract

Thanks to its ability to form duplexes through selective base-pair recognition, DNA is a
unique material for orderly self-assembled construction at the nanoscale. To develop a
nanotechnology platform on a grid of addressable molecular building blocks using DNA
node structures, DNA complexes need to be fixed onto surfaces. To fulfil this
requirement on lipid membranes, phosphoramidites monomers modified with a
cholesterol moiety and a spacer unit were synthesised. The hydrophobic spacer provides
separation between the hydrophobic cholesterol moiety and the phosphate backbone of
the DNA strand. For better anchorage of the network to a lipid surface, a hydrophilic
spacer was also used to link the cholesterol to the nucleoside. Solution studies demonstrated that the melting temperature (Tm) of the duplex with
adjacent cholesterols on each strand is much higher than that of the unmodified duplex.

The reliable and highly selective copper(I)-catalysed azide-alkyne 1,3-dipolar
cycloadition (CuAAC), the best known example of click chemistry, has proven to be of
remarkably broad utility in synthetic chemistry and in nucleic acid chemistry in
particular. CuAAC was exploited for the synthesis of a very stable double stranded
catenane duplex. The catenane was formed from a single stranded cyclic template and
its linear complement, using a third short oligonucleotide (ODN) as a helper for the
circularisation of the second ODN. The copper(I)-catalysed cyclisation of
oligonucleotides occurred by reaction between their terminal azide and their opposite
terminal alkyne, to produce a 1,2,3-triazole linkage between the two reactants. The
catenane was characterised by denaturing polyacrylamide gel electrophoresis, UV
melting studies and enzyme digestion.

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