ChemInform Abstract: DNA as Supramolecular Scaffold for Functional Molecules: Progress in DNA Nanotechnology

Abstract

Oligonucleotides have recently gained increased attraction as a supramolecular scaffold for the design and synthesis of functional molecules on the nanometre scale. This tutorial review focuses on the recent progress in this highly active field of research with an emphasis on covalent modifications of DNA; non-covalent interactions of DNA with molecules such as groove binders or intercalators are not part of this review. Both terminal and internal modifications are covered, and the various points of attachment (nucleobase, sugar moiety or phosphodiester backbone) are compared. Using selected examples of the recent literature, the diversity of the functionalities that have been incorporated into DNA strands is discussed.

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138 Chem. Soc. Rev., 2011, 40, 138–148 This journal is cThe Royal Society of Chemistry 2011
Cite this:
Chem. Soc. Rev
., 2011, 40, 138–148
DNA as supramolecular scaffold for functional molecules: progress in
DNA nanotechnology
Thomas J. Bandy, Ashley Brewer, Jonathan R. Burns, Gabriella Marth,
ThaoNguyen Nguyen and Eugen Stulz*
Received 22nd December 2009
DOI: 10.1039/b820255a
Oligonucleotides have recently gained increased attraction as a supramolecular scaffold for the
design and synthesis of functional molecules on the nanometre scale. This tutorial review focuses
on the recent progress in this highly active field of research with an emphasis on covalent
modifications of DNA; non-covalent interactions of DNA with molecules such as groove binders
or intercalators are not part of this review. Both terminal and internal modifications are covered,
and the various points of attachment (nucleobase, sugar moiety or phosphodiester backbone) are
compared. Using selected examples of the recent literature, the diversity of the functionalities that
have been incorporated into DNA strands is discussed.
1. Introduction
One of the most influential discoveries of the past century is
arguably the determination of the structure of double-
stranded DNA (dsDNA) by Watson, Crick, Wilkins and
Franklin.
1–3
Since then, the synthesis of DNA has reached a
level of sophistication where organic chemists can synthesise
almost any modified nucleotide and incorporate it site-
specifically into oligo-deoxynucleotides (ODNs). The well-known
structure of dsDNA (i.e. distinctive helicity, interior base-
stacking region, and major and minor grooves) allows for a
reasonable prediction of the structure of modified DNA
(Fig. 1). The selective recognition of the complementary strand
through the Watson–Crick base-pairing (AT and GC),
together with standardised and automated solid phase synthesis
using phosphoramidite building blocks, can be used to
specifically design new functional molecules. The modifica-
tions can be incorporated into the DNA strands at precise
sites, and the formation of the duplex will place the substi-
tuents in a predetermined and well-defined three-dimensional
arrangement. This may be outside of the duplex such as in
the major groove of the dsDNA, or within the hydrophobic
University of Southampton, School of Chemistry, Highfield,
Southampton SO17 1BJ, UK. E-mail: est@soton.ac.uk;
Fax: +44 (0)23 80 59 68 05; Tel: +44 (0)23 80 59 93 69
From left: Jonathan R. Burns, Ashley Brewer, ThaoNguyen
Nguyen, Eugen Stulz, Gabriella Marth and Thomas J. Bandy
Eugen Stulz received his PhD degree from the University of
Bern, Switzerland, for studies in the field of artificial nucleases
(Prof. Christian Leumann), and moved to Cambridge, UK,
as postdoctoral fellow in 1999 to work in supramolecular
porphyrin chemistry (Prof. Jeremy K. M. Sanders). In 2003
he moved to Basel where he held an independent position as
Fellow of the Treubel Fonds (Habilitation). In 2006 he was
appointed lecturer at the University of Southampton and was
promoted senior lecturer in 2010. His research interests are in
self-assembly of molecular systems based on (bio)molecules,
synthesis of nano-materials, and their applications in electronics
and medicine.
Thomas J. Bandy completed his undergraduate studies at the
University of Southampton, submitting his Masters thesis on
chiral Ruthenium and Osmium complexes under the supervision
of Prof. F. Richard Keene at James Cook University,
Queensland, before receiving his Masters degree in chemistry
in 2007. He then joined the research group of Dr Eugen Stulz at the University of Southampton, and is currently in the final year of his
PhD where his research interests include the templated assembly of supramolecular arrays of fluorophores and circular dichroism.
Chem Soc Rev Dynamic Article Links
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This journal is cThe Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 138–148 139
base-stacking interior. However, DNA structural details at the
site of modification are very difficult to ascertain and will not
be part of this review.
The reliability with which a specific DNA sequence
recognises its complementary sequence has been used in the
past ten years or so to create new nanometre-scale two- and
three-dimensional objects such as grids and lattices on
surfaces,
4,5
nanoscale patterns through folded DNA,
6
bipyramids,
7
cubes and cages,
8
all based on native DNA
strands.
9
Also RNA has been used as a building block in
nanotechnology.
10
Nano-structures using chemical modifica-
tions to introduce additional points of connectivity adds
further to the repertoire of available geometries.
11,12
Two-
dimensional DNA structures on surfaces can be used to
further position proteins or nanoparticles in order to form
grids that may be of use in diagnostics.
4,13
The commercial
availability of strand modifiers, in particular thiol end-
modifiers, allows for easy attachment of DNA strands onto
gold nanoparticles (AuNPs). With these techniques, different
AuNPs have been connected site-specifically using comple-
mentary DNA strands attached to the particles.
14
The utility
of novel DNA architectures in supramolecular chemistry,
biology and nano-technology clearly demonstrates the
versatility of this approach.
15,16
The combination of DNA
modification with a programmed architecture will certainly be
one of the future strengths of this approach, and complementing
similar efforts undertaken using peptide- or carbohydrate-
based scaffolds. Applications of functionalised DNA have
recently been reviewed separately, in particular, novel strategies
for site-specific DNA labelling,
17
chromophore labelling for
photoactive DNA-based nanomaterials,
18
assembly of chromo-
phores guided by nucleic acids
19
and biological applications of
conformationally restricted DNA analogues.
20
The introduction of modifications onto DNA can be
achieved via several different methods at various positions.
Sites available for modification include: 30- and 5 0-terminal
positions; 20- and 40-positions to the ribose ring; and finally,
modifications to one of the four natural bases, A, C, G, and T.
In addition, the nucleobase itself can be substituted with
designer molecules (artificial nucleobases), or the entire
nucleotide can be replaced with moieties that mimic the
function and structure of the DNA (base surrogates). These
modifications are discussed in the following sections in more
detail. Since a complete coverage of the literature in this
rapidly growing field of research is far beyond the scope of
this tutorial review, illustrative examples are selected to
demonstrate the versatility of the individual approaches.
In general, design of the modification and choosing an
appropriate methodology highly depends on strategic aims.
Fig. 1 Left: structure of B-type DNA duplex; right: A–T and G–C
base-pairs and numbered structures of the four nucleosides, deoxy-
adenosine, thymidine, deoxycytidine and deoxyguanosine.
Ashley Brewer studied as an undergraduate at the University of
Southampton and worked on undergraduate research projects
under the supervision of Prof. Fred Wudl at UCLA and UCSB;
he obtained his Masters in Chemistry in 2007. He joined Dr Eugen
Stulz’s research group at the University of Southampton in 2007
and is currently a final year PhD student working on porphyrin
substituted DNA arrays for use as supramolecular wires.
Gabriella Marth attended the University of Technology and
Economy in Budapest, Hungary, where she graduated in chemi-
cal engineering in 2005. She received her PhD degree in 2008 at
Sunderland University for her work on the synthesis of poly-
functional pyrroles and investigation of the chemoselectivity of
their reactions. After completing her doctorate she stayed at
Sunderland University for a postdoctoral fellowship to develop a
new synthetic route of natural product analogues under the
supervision of Prof. Paul W. Groundwater and Prof. Rosaleen
Anderson. In 2009, she joined Dr Eugen Stulz’s group as a
postdoctoral researcher working on a software-controlled
assembly of oligomers.
Jonathan R. Burns in 2007 received a degree in chemistry at the
University of Southampton which involved undergraduate
research with Prof. Tom Brown. Currently a final year PhD
student at Southampton University working with Dr Eugen Stulz
on energy transfer between porphyrins.
ThaoNguyen Nguyen studied Chemistry at University College,
Oxford University, and completed her M. Chem degree under
Dr Josephine M. Peach’s supervision. ThaoNguyen is currently
finishing her PhD thesis, titled ‘‘Porphyrin–DNA as Scaffold in
Nanotechnology’’, in the group of Dr Eugen Stulz.
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140 Chem. Soc. Rev., 2011, 40, 138–148 This journal is cThe Royal Society of Chemistry 2011
If the modification is to be attached via automated DNA
synthesis, then the building block has to be compatible with
standard DNA synthesis chemistry. In particular, compatibility
with the deprotection of the 4,40-dimethoxy trityl protection
group (3% TCA in DCM), oxidation (iodine solution), and
cleavage from the resin (conc. aq. NH
4
OH) may be limiting
factors. For 50-end modification, the protecting group can be
changed to avoid need for acid treatment. Post-synthetic
modification is an alternative strategy, which avoids these
issues, but may be restricted to a limited solvent range, such
as aqueous buffers, methanol, DMSO or DMF. Another
efficient method is use of PCR for the construction of longer
DNA duplexes than would be accessible by standard solid-
supported synthesis if the polymerase accepts the modifica-
tion, although producing low quantities of DNA. This method
requires the synthesis of 50-triphosphate nucleotides but does
avoid the use of reactive phosphoramidite monomers. The
major drawback of this methodology is that there is no control
over the site of incorporation of the modification in longer and
more complex DNA sequences.
2. Strategies to modify DNA at the 30-or
50-terminus
The list of commercially available end-modifiers for use in
phosphoramidite chemistry is growing steadily. Most
commonly, amino groups, thiol (disulfide) and carboxy
modifiers are being employed for further modification. Also
more unusual functional groups such as (masked) aldehydes,
acetylenes and azides (for click-chemistry) or halo-alkanes can
be attached to both termini (see Fig. 2 for a selection). Azide
modifications, however, need to be attached post-synthetically
to an amino-modified DNA. This part of the review focuses
on the most commonly used methods for DNA end-
functionalisation.
2.1. 50-Terminus modification
50-Modifications are usually achieved by one of two ways. The
first method is by reaction of a phosphitylated modifier, i.e. a
phosphoramidite building block, with the 50-hydroxyl group
of the final DNA base in the sequence. The modification is, in
this way, attached to the DNA strand via a phosphate group
and so forms an extension of the DNA backbone (Fig. 2). In
principle, any molecule with a hydroxy group available for
phosphitylation can be used and attached to the DNA
provided the rest of the modification is compatible with the
DNA chemistry.
The second method links the modification via direct
functionalisation of the 50-position of the ribose moiety prior
to coupling of the modified monomer. The chemistry may be
more flexible compared to the phosphitylation of modifiers.
The 50-position of thymidine, for example, is particularly
easily functionalised as protection of the nucleobase is not
normally required (as opposed to A, C and G). Examples
include ester formation, oxidation of the 50-OH to the
aldehyde for use in reductive amination, and transformation
of the 50-OH to an amine for amide formation. End-modification
also has the advantage that the DNA is much more tolerant, in
terms of structure and properties, of these substituents as there
are no steric constraints compared to internal modifications
(see below).
50-Terminal modifications have been studied for a variety of
reasons, but primarily to increase stability towards enzymatic
degradation, to increase thermal stability of the duplex
through capping, to enhance target affinity facilitating
detection (labelling) or for monitoring structural changes
(Fig. 3). One of the most comprehensive studies in terms of
end-capping substituents was reported by Richert et al., who
screened a total of 52 modifications, ranging from glycine to
vancomycin.
21
Berova et al. have used porphyrins as circular
dichroism (CD) markers to monitor structural changes in
DNA (Fig. 3).
22
The attachment of this achiral chromophore
to DNA places it into a chiral environment, and allows
excitonic coupling between porphyrin moieties giving rise to
characteristic CD spectra. These are very sensitive to the
porphyrin environment,
23
thus allowing, for example,
detection of the change from B to Z form of DNA.
22
It should
be noted that in addition to UV-Vis spectroscopy, CD spectro-
scopy is a major tool for analysis as it provides very useful
information about the DNA’s global structure.
Selective attachment of metals to the end of the DNA
has been achieved through ligand binding, allowing the
construction of metal templated frameworks and supra-
molecular assemblies.
24,25
By careful control of ligand design,
different metals can be selectively bound to the DNA
duplex and used, for example, to study charge transport
phenomena.
26
Using this approach, Sleiman et al.
27,28
were
able to bind a range of 3d transition metals selectively between
two duplexes containing either terpyridine or phenanthroline
Fig. 2 The top part shows a selection of commercially available
end-modifiers. CPG = controlled pore glass, DMT = 4,40-dimethoxy
trityl, PG = protecting group. Bottom: 50-modification through
tailor-made phosphoramidites (left) or substituted nucleotides (right).
Fig. 3 Principle of end-capping for increased DNA stability (left),
21
and attachment of a porphyrin marker for induced CD spectroscopy
(right).
22
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This journal is cThe Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 138–148 141
ligands (Fig. 4). For example, spontaneous oxidation of Cu(I)
to Cu(II) was observed when the metal ion was placed in the
incorrect environment for its optimum geometry, which was
reversed when the coordination environment was altered back
to the matching geometry. The site-specific incorporation of
metal centres into the DNA duplex also dramatically raises the
melting temperature with DT
M
=431C.
2.2. 30-Terminus modification
Standard automated oligonucleotide synthesis proceeds from
the 30- to the 50-end of the strand and as such a plethora of
affordable 30-phosphoramidites are commercially available.
The reverse 50-to3
0-end synthesis, which is the direction in
which the biosynthesis of DNA proceeds, is also possible.
29
However, the secondary 30-hydroxyl is less reactive than the
primary 50-hydroxyl and as such synthesis in this direction is
less efficient. Commercial 50-phosphoramidites also come at a
much greater cost than their 30-phosphoramidite counterparts
and thus this route is less popular. One general method for the
introduction of a modification onto the 30-end of DNA, whilst
avoiding costly 50-to3
0-synthesis, is to utilise standard
synthetic procedures, starting with a universal support.wAny
30-phosphoramidite, whether modified or not, can be attached
to a universal solid support. Subsequent DNA synthesis
proceeds from this, creating an oligonucleotide with the
modification at the 30-terminus of the strand. Letsinger
et al.
30
have demonstrated an example of this method of
synthesis by introducing a short phosphonite containing
linker directly onto the universal support. Oxidation of the
phosphonite to the monothio phosphate (Fig. 5) followed by
conventional 30-to5
0-synthesis, terminating in the intro-
duction of a 50-O-tosyl group at the 5 0-end of the strand,
allows for the formation of circular DNA after cleavage from
the solid support.
Meade et al.
31
have reported a method for attaching a
modification to the 30-end of the DNA strand by attachment
to the 20-position of the ribose moiety. The 2 0-position of a
ribose or deoxyribose ring is available for modification which,
if the modified monomer is used as the first base in a 30-to
50-sequence on a universal support, will place the modification
at the 30-end of the strand (Fig. 5). This approach allows the
30-end to carry out its role in the 30-to5
0-DNA synthesis, thus
avoiding the limitations of post DNA synthesis modification.
Meade’s approach was to condense a 20-amino nucleoside and
an aldehyde to yield a 20-imino modified nucleoside. The
resulting modification was a bidentate ligand (through nitrogen
lone pairs) which allowed for metallation by a ruthenium
bipyridine complex.
An alternative synthetic pathway to 30-modification was
demonstrated by Ihara et al.
32
In this case, 30-amino modified
DNA was synthesised by standard methods, and the strands
were deprotected and cleaved from the solid support before
coupling to an anthracene carboxylic acid for photo-induced
chemical ligation of DNA strands (Fig. 5). This post-DNA
synthesis coupling could be applied to any carboxylic acid
containing moiety, thus avoiding occasional problems during
phosphitylation and coupling of modified nucleobases. In fact,
direct addition of commercially available modifiers such as
amino, thiol and carboxy modifiers to a universal support
leads to the same methodology as in post-synthetic 50-end
modification and is usually the method of choice if modifica-
tions are to be incorporated here.
Fig. 4 Terpyridine and phenanthroline ligands used to alter the coordination environment on the 50-terminus, and to selectively and controllably
bind specific metals to the duplex.
28
Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
Fig. 5 30-End monothio phosphate, which can act as a nucleophile
(top left);
30
20-amino substitution for post-synthetic functionalisation
through imine-formation (top right),
31
30-amino modified DNA
for introduction of carboxylic acid substituted functional groups
(bottom).
32
wThe standard controlled pore glass (CPG) solid supports have the
first nucleotide already attached to the beads. A universal support
comes ‘‘unloaded’’, i.e. there are no nucleotides or other modifiers
attached to the CPG, thus any phosphoramidite that is loaded onto
the universal support will inevitably form the 30-end of the DNA
strand.
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142 Chem. Soc. Rev., 2011, 40, 138–148 This journal is cThe Royal Society of Chemistry 2011
3. Artificial nucleobases for internal DNA
modification
Another category of modifications to DNA include
positioning moieties within the base-stacking region of double-
stranded DNA. Modifications of this type are replacements
for various parts of the DNA structure, be it artificial nucleo-
bases attached to the (deoxy)ribose, or backbone modifica-
tions with complete replacement of the nucleoside. As outlined
in the Introduction, one of the main properties that makes
DNA such an attractive medium for manipulation and
modification is its facile stepwise synthesis and the specificity
and strength of duplex formation. This also means that
modifications can be made to the nucleosides in the centre of
the strand whilst retaining the preorganisation due to the
natural Watson–Crick base pairs either side of the modified
section (flanking sequences).
3.1 Artificial nucleobases
The removal of a natural nucleobase and replacement with an
artificial one allows access to a wide range of modifications
(Fig. 6). Most artificial nucleobases are planar aromatic
compounds, which are able to stack within the duplex. These
can be designed, either to form non-covalent interactions
between the two strands, or to stack on top of each other in a
zipper-like fashion, partly relying on the strength of the inter-
action between natural base pairs elsewhere in the duplex. By
removing the conventional nucleobase the choice of artificial
building blocks is limited only by the chemistry of attachment
to the ribose ring, stability to phosphoramidite or ODN synthesis
conditions, size and the imagination of the chemist.
A large variety of nucleosides with artificial nucleobases
have been synthesised, including but not limited to phen-
anthroline, naphthalene, stilbene, pyrene, coumarin, terphenyl,
biphenyl, bipyridine and porphyrin, most of which have been
covered in a review by Kool.
33
Examples of conventional base
analogues are also included in this review. The aromatic
nature of the artificial nucleobases is crucial for obtaining a
stable duplex through p-stacking, although the dsDNA is
remarkably flexible, tolerating and accommodating artificial
bases that are not perfect mimics of the natural bases. The
thermal analysis and structural solution by NMR spectro-
scopy of multiple bi-phenyl modified dsDNA by Leumann
demonstrates for the first time that interstrand stacking can
increase duplex stability,
34
and hydrogen bonding between the
natural bases is not crucial in these systems.
35
Multiple inser-
tions of these base-surrogates lead to a zipper-like arrange-
ment (Fig. 6).
36
Such modifications are very attractive as
fluorescence markers; the fluorescence is normally quenched
to a great extent when encapsuled in a perfect DNA duplex,
but is retained to a large portion in a non-ideal duplex
environment, e.g. when bulges or mismatches are present in the
flanking sequences. A remarkable exception is a binaphthyl
nucleoside recently reported by Seitz,
37
which shows an increase
in fluorescence upon multiple incorporation into DNA.
Other more recent advances by Kool et al.
38
describe
mimicking the conventional base-pairs using expanded ring
structures, denoted yDNA bases (Fig. 7). Despite their
increased size, the novel nucleobases can be recognised and
replicated in vivo by natural polymerases, and have also been
Fig. 6 Examples of artificial nucleosides
33
and schematic representation of zipper-like inter-strand stacking within the DNA duplex.
Fig. 7 Comparison between natural Watson–Crick base-pairs AT
and GC (top), and the analogous ‘yDNA’ base pairs AyT and GyC
(bottom).
38
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This journal is cThe Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 138–148 143
successfully replicated within bacterial cells. This example also
very nicely demonstrates that the use of artificial nucleobases
is not only restricted to the synthetic laboratory for use in
supramolecular chemistry, but may well have applications in
synthetic biology for expanding the genetic alphabet.
3.2 Binding of metal ions
Artificial nucleobases can also be used to chelate metal ions.
The natural hydrogen-bonded base pair is completely replaced
by a ligand–metal–ligand analogue that forms a non-covalent
bond across the DNA duplex, binding the two strands
together. This has the advantage that a chain of metals can
be built up within the double helix itself, which has interesting
applications in energy transfer and nanowire formation, as the
exact composition of the assembly can be controlled precisely.
Examples of metal-controlled ‘‘base-pairs’’ include the
pyridine-2,6-dicarboxylate and pyridine-2,6-dicarboxamide
nucleobases, each of which form a Cu(II)-mediated base pair
with a pyridine nucleobase on different strands (Fig. 8a). By
replacing a single base pair in the middle of a 15-mer sequence,
Schultz et al.
39
have demonstrated an increased stability of the
duplex which is manifested by an increase in the melting
temperature T
m
compared to natural DNA.
An extension of the concept of using artificial metal-binding
nucleobase is hydroxypyridone as ligand.
40
This initial system
was expanded by Shionoya and Carell
41
to engineer a system
including the hydroxypyridone nucleobase as one of two
different metal-complexing units (alongside a pyridyl nucleo-
base), to selectively produce an oligonucleotide double helix
with a string of complexed metal ions in replacement of the
standard base pairs (Fig. 8b). Two hydroxypyridone units on
adjacent strands form a planar ‘‘base pair’’ on complexation
with Cu(II) ions, whilst two pyridyl units complex an Hg
2+
ion. It was also demonstrated that thymine–thymine
mismatches can selectively bind Hg(II) ions in the system, with
standard DNA as flanking sequences to aid preorganisation.
Cu(II) and Hg(II) ions can then be selectively bound in the
desired sequence dictated by the sequence of cation binders,
leading to a programmed assembly of a hetero-metallic nano-
wire. The system was recently expanded to self-assemble a
triplex around octahedrally coordinated Fe(III) ions.
42
A
triplex containing four Fe(III) base triplets was synthesised,
and so demonstrates the extension of this technique to allow
octahedrally-coordinated transition metals to be incorporated
into metal–oligonucleotide complexes and nanowires.
3.3 Backbone modifications and nucleotide replacement
A related type of modification is the complete replacement of a
section of the oligonucleotide. Such a modification would
require flanking sequences of natural dsDNA to maintain
the helical DNA structure. Again, any diol-containing
functional group can be used to form a building block, where
one of the hydroxy groups is DMT-protected while the other
one is transformed into the phosphoramidite. The use of
aromatic replacement units is preferred for reasons
mentioned above.
Ha
¨ner et al.
43
have synthesised an oligomeric DNA strand
with a central section consisting of up to seven pyrene
modified subunits (Fig. 9). The system is self-organising and
forms a duplex with the formation of an interstrand helical
stack of pyrene subunits. Despite the fact that pyrene subunits
are planar and achiral themselves, they adopt a right-handed
helix which is imposed on the system by flanking natural DNA
sequences. Other mixed DNA hybrids have been synthesised
using tetrathiafulvalene, perylene diimide and phenanthrene
modifications.
44
Analogously, the ribose can be replaced with a short alkyl
chain having a perylene unit on a side chain as described by
Wagenknecht et al.
45
The short alkyl chain mimics the
distance between adjacent phosphates in natural oligonucleotides
(Fig. 10). Each modified perylene unit is placed opposite an
Fig. 8 (a) A Cu(II) mediated metallo-base-pair.
39
(b) Schematic
representations of Cu(II) and Hg(II) mediated metallo-base-pairs.
41
The metal ions reside within the double helix itself, and the sequence
can be precisely controlled during the DNA synthesis.zReprinted by
permission from Macmillan Publishers Ltd:
41
copyright 2006.
zWe believe that the right-hand DNA strands depict the incorrect
directionality of the DNA. The figures were redrawn or reprinted from
the corresponding publications.
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144 Chem. Soc. Rev., 2011, 40, 138–148 This journal is cThe Royal Society of Chemistry 2011
abasic site on the adjacent strand, and the perylenes are
positioned alternately to form a zipper-like interstrand
sequence. Again, this adopts a right-handed helix with the
presence of natural Watson–Crick base pairs at both termini.
By this method, organic aromatic molecules can be arranged
into a helical array where the supramolecular structure is
predetermined by the DNA.
The backbone modification described by Sheppard
29
includes the synthesis of two natural oligonucleotide strands,
one with salicylaldehyde units at both the 30- and the
50-terminal position. Addition of the complementary strand
aligned the two salicylaldehyde units in adjacent positions, and
addition of a square planar metal ion, such as Mn(II) or Ni(II),
was used to ligate the two sequences (Fig. 10). The metal ion is
removed and the result is two strands ligated about a central
spacer. Such a system could be expanded to allow facile
connection of multiple oligonucleotide strands; however, it is
metal-templated DNA synthesis that is very promising for the
synthesis of novel biomolecules. This system has recently been
extended by Brown
46
to the use of copper-catalysed azide–
alkyne cycloaddition (CuAAC) reaction which is the best
example of click-chemistry. The tolerance of the triazole
linkage by polymerases suggests great potential of this system
in bio-organic chemistry.
4. DNA containing external modifications
From a supramolecular view point, DNA can provide a
versatile scaffold for attachment of functional molecules
e.g. porphyrins,
47
pyrenes,
48,49
metallated bipyridines or
terpyridines
50,51
and various other alkyl and aryl
substituents.
52
The substituents which are attached to the
outer rim of the DNA form a helical array upon hybridisation.
Modifications can be located in the major or minor groove of
DNA, the former being the more commonly chosen site of
attachment since the major groove is larger and can accom-
modate bulky substituents better, but also for synthetic
reasons as will be shown by the examples discussed below.
All nucleobases are commercially available as their
iodinated analogue. However, by far the most commonly used
starting nucleobase is 5-iodo-20-deoxy uridine (5-I-dU),
primarily because thymidine (and dU) do not normally require
protection of the nucleobase, and the 5-I-dU is the most cost-
effective starting material. Modification of iodinated bases by
Sonogashira cross-coupling
53
or by Stille cross-coupling
54
is
facile and allows for various different substituents to be
attached to the DNA. The majority of modifications are
attached to monomers prior to DNA synthesis. Alternatively,
Richert has demonstrated that Sonogashira coupling to an
iodinated single-strand whilst on the solid support is possible
for a wide variety of substituents.
52
If the site of functionalisation on the nucleobase is chosen
appropriately, the modification will protrude from the double
helix into the major groove, whilst the base pairs still interact
via Watson–Crick hydrogen bonding. This geometry does not
Fig. 9 Schematic representation of oligopyrene repeat units flanked by DNA double-helix sequences.
43
Copyright Wiley-VCH Verlag GmbH &
Co. KGaA. Reproduced with permission.
Fig. 10 Structure of perylene subunit (left) as base surrogate;
schematic view of DNA double helix (middle) with central nucleobases
replaced with alternating perylene moieties;
45
ligated DNA strand
obtained by DNA templated metal compex formation (right).
29
Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with
permission.
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This journal is cThe Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 138–148 145
seem to distort the helix to a large extent. To maintain the
helical structure and the base pairing, pyrimidines should be
modified at the 5-position, and purines should be replaced with
7-deazapurines with modifications made at the 7-position. An
example by Famulok et al.
55
shows that all nucleotides in a
DNA strand can be modified with functional groups which are
derived from amino-acid side chains (Fig. 11). These are
suitable substrates for polymerases, thus a fully functionalised
DNA can be amplified via PCR. That even larger substituents
are tolerated by polymerases was shown early on by Seela.
56
The use of cross-coupling reactions of nucleoside
triphosphates and consequent use in PCR has been reviewed
recently by Fojta and Hocek.
57
Theirworkalsoconfirmedthat
modification at the 7-position of a 7-deaza purine-50-O-
triphosphate is preferred over the 8-position. This is because
the latter provide poor substrates for DNA polymerases due
to enhanced steric hindrance in the DNA backbone.
50
The
cross-coupling approach involves direct attachment of
metallated [Ru(bpy)
3
]
2+
and [Os(bpy)
3
]
2+
acetylenes onto the
nucleobase-triphosphates (Fig. 11). The Ru(II) and Os(II)
bipyridine metal complexes were attached to all four bases,
and the building blocks were incorporated into DNA using
PCR to enable sequence-specific incorporation of the metal
complexes.
56
Ru(II) and Os(II) based chromophores have also been used
for photophysical studies whereby the chromophores exhibit
donor- and acceptor-type interactions, respectively. In
particular, Tor et al.
58
have attached [(bpy)
2
Ru-3-ethynyl-
1,10-phen]
2+
and [(bpy)
2
Os-3-ethynyl-1,10-phen]
2+
chromo-
phores rigidly via an acetylene bond to dU. Systematic
variation of the distance-separation between chromophores
revealed quenching of the Ru-complex’s fluorescence which
was approximately proportional to the Fo
¨rster dipole–dipole
mechanism (Fig. 12). Notably, they also discovered that
Fig. 11 Modified nucleoside-50-O-triphosphates as substrates for PCR mediated DNA synthesis of fully modified functional DNA (top)
55
and of
metal complex functionalised DNA (bottom).
50
Fig. 12 Base-pair separation of chromopores vs. FRET efficiency based on emission quenching of a ruthenium complex. Reprinted with
permission from ref. 58. Copyright 2002 American Chemical Society.
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146 Chem. Soc. Rev., 2011, 40, 138–148 This journal is cThe Royal Society of Chemistry 2011
changing the rigid acetylene linker to a flexible dimethylene
linker, the donor–acceptor pairs revealed a greater correlation
with Fo
¨rster type behaviour. Synthesis of these strands was
achieved using standard solid-phase DNA techniques,
analogously to what was reported by Grinstaff
51
for bipy-
complexes attached via a propargyl-amide linker. This also
demonstrates that the use of solid-phase DNA synthesis is a
better method to control precise positioning of functionalities
than PCR.
The examples which have been described above demonstrate
how DNA can be modified externally by different chemical
functionalities, upon which a variety of chromophores can be
attached which may potentially have applications in redox and
fluorescence labeling, drug delivery and nanotechnology; the
latter potential was laid out in a feature article by Wengel.
59
Indeed it is this application that has attracted major interest,
and DNA is being used as a scaffold to create photonic and
electronic wires. Recent work by Wagenknecht,
48
Stulz,
60
Wengel,
61
Schuster
62
and Nakamura and Yamana
63
showed
that fluorophores can be covalently connected to DNA and
RNA and aligned within the grooves to form a helical array of
stacked chromophores (Fig. 13). Again, most commonly used
sites for modification are the 5-position of dU
48,60
or C,
62
the
20-hydroxy group of the ribose,
64
or 20-amino modified LNA
building blocks.
61
It should be noted that attachment of the
modifications to the 20-position of the ribose will direct the
modifications into the minor groove of the DNA, as compared
to attachment onto the nucleobase.
The stability of the resulting array depends on the design of
the system, i.e. if the chromophores are attached to one strand
only, or if they are attached to both complementary strands in
an alternating manner. The latter leads to external zipper-
arrays (see also 3.1 for the discussion on internal zipper-
arrays). For example, zipper-like stacking of pyrenes does
not greatly reduce the stability of the duplex, and it is certainly
an advantage if the integrity of the system is maintained.
48
Hydrophobic substituents such as porphyrins tend to
destabilise the DNA duplex significantly if attached to one
strand only, despite the indication that the structure is not
greatly altered as shown by CD spectroscopy and molecular
modelling.
47
However, attachment of porphyrins onto
complementary strands forms a zipper-like arrangement with
enhanced duplex stability, most likely due to p–p-stacking and
hydrophobic interactions. The porphyrins have the advantage
that they can be metallated without disturbing the dsDNA,
thus the reversible formation of potential photonic wires based
on metal complexes becomes possible.
65
Another strategy to efficiently assemble porphyrin–DNA
structures was followed by Seeman and Majima. Herein a
maleimide substituted tetraphenyl porphyrin was conjugated
with four thiol functionalised DNA strands. Upon duplex
formation with complementary strands, four double helices
were assembled and used to create porphyrin diads, showing
efficient energy transfer from a Zn porphyrin to a free base
porphyrin (Fig. 14a).
66
Potentially, this DNA structure could
accommodate host–guest system within the cofacial porphyrin
dimers. This four-way-branched DNA was also used as a
Fig. 13 Comparison of the calculated structures of pyrene–DNA,
49
porphyrin–DNA,
65
pyrene–RNA,
63
benzoyl-LNA
61
and aniline–DNA
62
(from left to right).y
Fig. 14 (a) Four-way-branched porphyrin–DNA assembly (Reprinted with permission from ref. 66. Copyright 2005 American Chemical Society);
(b) use of porphyrin–DNA in a tile system to create DNA tubes (Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with
permission).
67
y(a), (b) Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Repro-
duced with permission. (c), (d) Reproduced by permission of the Royal
Society of Chemistry. (e) Reprinted with permission from ref. 62.
Copyright 2008 American Chemical Society.
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This journal is cThe Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 138–148 147
connector in a DNA tile system to build DNA nano-tubes
(Fig. 14b).
67
These examples illustrate that DNA as a
supramolecular scaffold provides an excellent backbone for
external covalently linked functionalities, ranging from opto-
electronically active chromophores to organic polymers such as
nylon, as shown by Seeman who described a nylon/DNA
ladder polymer.
68
In general, chromophores show strong electronic inter-
actions when attached to DNA, and energy transfer can be
achieved along the DNA with the appropriate design of the
array, as shown by Tinnefeld.
69
A recent example by Brown
and Norden further demonstrates the suitability of DNA
based nano-architectures for the creation of artificial photo-
synthetic systems.
70
In this example, a DNA based assembly of
a light-absorbing antenna (fluorescein) and a redox switch
(porphyrin) were anchored onto a lipid membrane. Light-
induced energy transfer from the fluorescein to the porphyrin
triggered electron transfer from the porphyrin to a membrane
based quinone derivative. The excitation energy is therefore
trapped in the lipid phase of the membrane in the form of a
radical anion, which might be used for further chemical
reactions.
5. Conclusions
The growing diversity of modifications which are available for
functionalising DNA whilst maintaining its integrity is a major
step forward in DNA nanotechnology. In particular,
incorporation of metal complexes and chromophores for the
creation of photonic or electronic wires is reaching a level
beyond proof-of-concept and is under investigation to demon-
strate their utility in energy or electron transfer. Future
applications are to be expected in the fields of optoelectronics,
diagnostics, therapeutics and possibly in catalysis. The next
step forward will be to combine the many different types of
modifications to create designer molecules that have all desired
functionalities. Taking the synthesis beyond the short DNA
strand, and incorporating this concept into sophisticated
three-dimensional DNA nanostructures, such as cubes or
tetrahedra, or into structured arrays on surfaces, will
ultimately lead to a very large diversity of new DNA-based
functional assemblies. Some existing examples have been
presented here, and a review by Endo and Sugiyama on the
chemical approaches to DNA nanotechnology highlights the
recent achievements towards this goal.
71
Together with other
approaches, which are based on natural templates such as
peptides or saccharides, this field of research will have a great
influence in the way future materials will be designed.
Certainly, today nucleic-acids chemistry is more than just
automated solid-phase DNA synthesis, but without this
invention
72–74
the current achievements would not have been
possible.
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