Since the discovery in the early 1980s that RNA was
not simply a passive carrier of genetic information but
could participate directly in catalysis in living cells1,2,
our understanding of RNA structure and function
has been in constant flux. Another important insight
emerged in 1990 when three separate groups used
in vitro selection approaches to isolate RNA and DNA
molecules that bind tightly to several nucleic-acid
binding proteins, and to organic dyes used for affinity
The isolation of novel RNAs that bind to small
organic molecules confirmed the assumption — based
on the ability of RNA to fold into complex, three-
dimensional shapes rivalling those of proteins — that
in degenerate sequence libraries of ≥1015 molecules,
which are easily created and manipulated in the
laboratory, there is probably an RNA molecule with
a fold to bind almost any target5. A logical extension
of this idea was that aptamers that bind transition-
state analogues that mimic the chemical structure of
key intermediates in enzyme catalaysis would also be
catalytically active, analogous to catalytic antibod-
ies. This has turned out to be true even for complex
organic reactions6,7. These results provide compelling
experimental evidence in support of the ‘RNA world’
hypothesis, which postulates that the biological world
evolved from a self-replicating RNA molecule that was
assembled by chance8.
An iterative protocol for the in vitro selection experi-
ments discussed above4 was introduced and called
‘systematic evolution of ligands by exponential enrich-
ment’ (SELEX) (FIG. 1). The products of the selection5
were called ‘aptamers’ (from the Latin aptus meaning
‘fitting’). By analogy with antibodies the targets bound
by aptamers were called ‘apatopes’, although ‘epitope’
or ‘target’ is the most frequently used terminology. The
selection protocol has subsequently been reduced to
an automated in vitro process9–11, opening the way for
high-throughput selection against an almost infinite set
of targets. The range of aptamer reagents isolated can be
assessed by reference to an online database.
Given the biological significance of aptamer– apatope
interactions, it seemed unlikely that Nature had
neglected this field of biomolecular recognition and
indeed over the past few years we have learned about
naturally occurring riboswitches12,13. These allow regu-
lation of gene expression by changes in the conforma-
tion of mRNA transcripts in response to alterations in
physiological conditions, mediated by the binding of
small molecular-weight metabolites within an ‘aptamer
domain’. These mRNA conformational changes effect
translational or transcriptional termination, or even
self-cleavage (BOX 1).
Commercial exploitation of the aptamer field has
lagged behind research discoveries. However, last year
the first aptamer-based therapeutic agent (Macugen)
for treating a form of macular degeneration entered clini-
cal use14–16. So, on the sixteenth anniversary of the first
publications one can argue that aptamers have finally
come of age. Here we discuss significant recent develop-
ments in the field of aptamer research, and draw atten-
tion to work aimed at developing new aptamer-based
Structural lessons from aptamers
Aptamers selected against nucleic-acid binding pro-
teins and small molecular-weight ligands have been
used to investigate protein and nucleic-acid sequence
specificity. The Gold laboratory showed that a natural
RNA stem-loop sequence is one of two distinct but
related sequences that can bind bacteriophage T4 DNA
polymerase with almost equal affinity4. Therefore, the
‘sequence space’ for such interactions contains several
combinations that give rise to mutual binding affinity,
and potentially there are combinations that have not
been exploited during evolution.
Astbury Centre for Structural
of Leeds, Leeds LS2 9JT, UK.
Correspondence to P.G.S.
A condition in which the light-
sensing cells of the macula,
which is in the centre of the
retina, malfunction and cease
to work, leading to reduction or
loss of central vision. The
disease can be caused by the
leakage of newly forming blood
vessels into the retina and it is
this process that is susceptible
to treatment by Macugen.
All the possible sequence
combinations in a nucleic-acid
library used for SELEX. As
there are many such
sequences, it is unlikely that all
possible combinations of
sequence and function have
been ‘tried’ during evolution.
Aptamers come of age – at last
David H. J. Bunka and Peter G. Stockley
Abstract | Nucleic-acid aptamers have the molecular recognition properties of antibodies, and
can be isolated robotically for high-throughput applications in diagnostics, research and
therapeutics. Unlike antibodies, however, they can be chemically derivatized easily to extend
their lifetimes in biological fluids and their bioavailability in animals. The first aptamer-based
clinical drugs have recently entered service. Meanwhile, active research programmes have
identified a wide range of anti-viral aptamers that could form the basis for future therapeutics.
588 | AUGUST 2006 | VOLUME 4
© 2006 Nature Publishing Group
is incubated with
Nucleic acids are amplified
2′-methyl or 4′-thio
Low affinity species
Bound species are eluted
Surface plasmon resonance
(SPR). A technique for
monitoring the affinity between
molecules in solution (analytes)
as they pass across an
immobilized target on the SPR
sensorchip. In aptamer
research, this technique is used
to collect slowly dissociating
aptamer species that have
higher affinity than those that
A term derived from the
German word for mirror. These
are RNA aptamers synthesized
chemically with L-ribose
instead of the natural D-ribose
and are therefore resistant to
A similar series of experiments were carried out
on the translational repression complex that forms
between a 19 nucleotide RNA stem-loop operator in
the genomic RNA and a dimer of the coat protein in the
RNA bacteriophage MS2. The translational repres-
sion complex is used as a model for understanding
sequence-specific RNA–protein interactions. SELEX
showed that the natural RNA stem-loop operator
binds tightly to the phage coat protein17, although an
RNA aptamer that differs from the consensus opera-
tor by a single nucleotide emerged from this analysis
as the tightest binder. Slightly reducing the selection
pressures in such experiments, or introducing muta-
tions into the RNA-binding site on the coat protein,
yielded aptamers with sequence variants that seemed
to break previously identified consensus rules for
RNA stem-loop–protein binding18. However, X-ray
structure determination of these novel aptamer–
protein complexes19,20 showed that the aptamers still
bound at the natural RNA-binding site, and that the
crucial elements in the aptamer consensus sequence
simply needed to be adjusted to reflect the structural
constraints in this system, which are the number of
nucleotides between adenines that are bound to the
protein (FIG. 2).
Similarly, for DNA-binding proteins, the sequences
of natural binding sites, such as operators, have been
selected by evolution in the context of many DNA-binding
proteins. To avoid accidental cross-binding to the same
regulatory site by different transcription factors, selected
sites need to be discriminated efficiently, rather than being
the tightest binders. Indeed, as such interactions need to
be reversible to allow gene regulation, the tightest possible
binding sequences are often not found in vivo but emerge
from in vitro SELEX experiments21.
Interestingly, when aptamers are selected against small
molecular-weight ligands that bind natural RNA targets,
such as aminoglycoside antibiotics, a wider range of binding
sequences is revealed22,23. Structural studies show that these
aptamers often present unusual binding surfaces, which
these ligands fit into; however, this does not necessarily
correspond to the biological interaction24,25. Structures
of selected ribozymes, such as the Diels–Alder ribozyme,
which catalyses the formation of two carbon–carbon bonds
in a reaction of general importance in organic chemistry,
also reveal unusual ways in which reactants can be bound
to promote catalysis26. Obviously it is also possible to select
aptamers against targets that do not normally bind nucleic
acids, and this possibility extends the potential functions
and applications of nucleic acids into new realms.
Recent technical innovations
The basic principles of SELEX have remained largely
unaltered since its conception (FIG. 1). Considerable
improvements have, however, been made to the selection
step and to the properties of the resulting aptamers.
Most advances in aptamer isolation have aimed at
improving the efficiency of selection, that is, reducing the
number of cycles or the time taken to isolate high-affinity
species. The Ellington group was the first to automate this
process, reducing the isolation time from several months
to a few days9–11. Other groups have focused on improving
partitioning efficiency. High-affinity aptamers have been
isolated using surface plasmon resonance (SPR) by fraction-
ating the dissociation phase of the analyte-binding reac-
tion, thereby isolating only those species with the slowest
‘off-rates’ (REFS 27,28). High-affinity DNA aptamers have
also been isolated in a single round using capillary elec-
trophoresis to separate free protein or nucleic acids from
complexed material29. Other key areas of improvement
are in aptamer biostability and bioavailability. The ability
of T7 RNA polymerase to incorporate 2′-amino-modified
or 2′-fluoro-modified pyrimidines30,31 allows isolation
of aptamers with greatly improved biostability. More
recently, an evolved RNA polymerase was reported that
could efficiently incorporate 2′-methyl pyrimidines into
transcripts, which also results in increased biostabil-
ity32. The 4′-thio pyrimidines have also been added to
this family of RNA-stabilizing nucleotides33. An elegant
approach involves the use of l-ribose-based nucleotides
to generate biostable Spiegelmers34 following selection
of natural d-ribose sequences against the enantiomer of
the biological target. Also, 3′–3′ linked dinucleotide caps
and circularization or disulphide crosslinking35 have
been shown to improve aptamer stability, although these
modifications are less commonly used.
Figure 1 | Basic outline of a single SELEX round. a | A degenerate nucleic-acid
sequence library is incubated with the target molecule under defined solution
conditions. b | Target-bound nucleic acids are partitioned. c–e | Species with
lower binding affinity are removed and the bound species are eluted, allowing
preferential amplification of higher affinity species. This enriched pool is then used as the
starting point in subsequent cycles. Typically, 10 to 20 cycles are carried out before
aptamer characterization. In early rounds, species with no affinity are competed out of
the pool. In later rounds, molecules with affinity compete for binding sites on the target.
Such competition results in enhancement of the pool binding-affinity in a manner similar
to Darwinian evolution. Recent technical developments described in the text are listed
alongside each step in brackets. CE, capillary electrophoresis; SELEX, systematic
evolution of ligands by exponential enrichment; SPR, surface plasmon resonance.
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Two molecular structures that
have identical chemical
compositions but are non-
superimposable in three
dimensions — they are mirror
images of each other. For
amino acids and ribose sugars
these are known as the D- and
The bioavailability of aptamers is a significant issue
that affects their application as in vivo diagnostic and
therapeutic tools. The most common approaches
to improve bioavailability include surrounding the
aptamer with lipoproteins36 or the attachment of bulky
groups, such as polyethylene-glycol36–38, cholesterol39
or biotin–streptavidin40, to the 5′ or 3′ ends, resulting
in reduced renal clearance and therefore an increased
plasma half-life. Aptamers that bind intracellular
targets require either targeted delivery or recombinant
expression, each of which has associated technical
Variations of the SELEX protocol have allowed
isolation of aptamers with specific desired proper-
ties, expanding the repertoire of aptamer func-
tions. Toggle-SELEX, for example, allows isolation
of aptamers with a broader range of specificities by
selecting against related targets in alternating cycles41.
Box 1 | Riboswitches — Nature’s aptamers
Applications of artificial RNA ligand-binding
domains were fairly advanced90 before it was
realized that such interactions are biologically
relevant and are used to regulate gene
expression. Riboswitches are rapidly joining
anti-sense and small interfering RNAs as
important biological tools13,91. It is now known
that regulated conformational changes in
mRNAs in response to temperature92 or ligand
binding lead to translational or transcriptional
regulation. Ligand binding can even drive
RNA to fold into an auto-cleaving ribozyme93.
Discoveries about riboswitches are now
being exploited actively to develop new
gene-expression control and selection
systems, including applications of
recombinant ribozymes94 and aptazymes95.
Aptazymes are allosterically regulated
ribozymes. Novel engineered ribo-
regulatory elements that allow artificial
combinations of ligand-binding domains
and allosterically regulated RNA
conformational change to control gene expression have also been described96,
as have bioinformatics approaches to identify functional ribo-domains in natural
transcripts97. These approaches will allow development of ‘controllable gene
knockouts’, which could provide information about the temporal nature of a
gene’s activity more readily than traditional knockouts98.
An exciting development is the use of synthetic riboswitches to detect binding
of small molecular-weight ligands in vivo. For instance, the introduction of the
anti-theophylline aptamer into the 5′ UTR of a β-galactosidase reporter in Escherichia
coli induced increased expression of β-galactosidase on addition of theophylline, but
not the closely related caffeine, to the culture medium99. The authors had anticipated
the opposite effect — they had assumed that theophylline binding would ablate
β-galactosidase expression by obstructing ribosome binding. However, the effect was
highly specific and relatively unaffected by changes to the position of the aptamer
sequence. Modification of this approach allows a range of applications for molecular
evolution studies in vivo.
The molecular basis for these remarkable RNA-based regulatory systems is now
beginning to be understood at the atomic level, as a result of several recent X-ray
crystal structures26,100. The figure, part a, shows a summary of the results from X-ray
crystallography of the adenine-bound form of the Bacillus subtilis add A-riboswitch.
The added base (shown in red) is recognized at every hydrogen-bonding position by
interactions with three bases from the riboswitch. This explains the discrimination
against guanine binding. Hydrogen bonds are shown as dotted lines, and the blue
circle denotes a water molecule. The structure of a Diels–Alder ribozyme with and
without bound product has also been solved. The λ-shaped ribozyme creates a three-
dimensional fold that closely matches the shape of the transition state of the reaction,
explaining the catalysis26.
The B. subtilis add A-riboswitch is converted by addition of the base adenine from a
translationally repressed state, in which the Shine–Dalgarno (SD) sequence (shaded red)
and the initiation codon (shaded yellow) are sequestered by pairing interactions, to an
activated state, where these segments are free to interact with the ribosomal and tRNA
(see figure, part b). Figure modified with permission from REF. 100 © (2004) Elsevier.
590 | AUGUST 2006 | VOLUME 4
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Tailored-SELEX involves ligation and cleavage of
primer sites before and after amplification42, allowing
the isolation of shorter aptamer sequences that are
more readily synthesized chemically. Photo-SELEX
involves UV-induced crosslinking of aptamers con-
taining light-sensitive nucleotides to their targets,
greatly increasing binding affinity43.
Applications of aptamers
The applications of aptamers are so numerous that
studies describing their use appear in the literature
on a weekly basis. The versatility of this technology is
reflected in the fact that there are few areas of research
to which aptamers cannot be applied. Some examples of
their expanding use are outlined below.
Figure 2 | Structural lessons from RNA aptamer–protein complexes. The X-ray structures of the translational
repression complex formed by the MS2 coat-protein dimer and the natural stem-loop operator of 19 nucleotides (top left),
and the equivalent complex with the consensus aptamer sequence (F6) from a SELEX experiment (top right)18,20 are
illustrated. The coat-protein polypeptide backbones are shown as gold and blue ribbons with the RNAs as orange stick
models covered by space-filling surface representations. Note the overall similarity of the complexes formed in each case,
including the fact that the aptamer binds to the natural stem-loop interface on the protein. Below are representations of
the intermolecular contacts made in each case. Hydrogen bonds between the MS2 protein and RNA are shown as dotted
lines, the two adenosines that are tightly bound by the protein (A–10 and A–4) are indicted in bold. It was believed that a 4
base loop was an essential feature of the operator consensus but F6 has a 3 base loop. The loops are highlighted in red in
each case. The number of nucleotide steps between the adenosines at positions –10 and –4 (numbers relative to the
replicase start codon) seem to be more important and these bases have been coloured yellow in each case. Water
molecules are shown as blue ovals.
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A collection of separation
techniques, which involve the
application of high voltages
across buffer-filled capillaries
to achieve separations based
on a range of different physical
Purification and biotechnology. One of the most obvious
uses for highly specific, high-affinity, reusable molecules
is as an affinity purification medium. An advantage of
aptamers over most other solid media derivatized with
the affinity target is that the pure protein can be obtained
in fewer steps, owing to the ability of aptamers to dis-
criminate between closely related ligands. Furthermore,
aptamers can be selected to bind to the natural form of
the protein, eliminating modifications with tags (such
as glutathione S-transferase (GST) and His) which can
adversely affect protein folding, structure and function.
Subsequent tag-cleavage steps that often reduce yields
are also no longer necessary. An early example of this
approach used an immobilized anti-selectin aptamer to
purify a selectin-receptor globulin expressed in Chinese
hamster ovary cells44. A 15,000-fold purification of the
fusion protein in a single step with 83% recovery was
reported. More recently, the discriminatory ability
of aptamers was demonstrated by the purification of
d-arginine-vasopressin (dissociation constant (Kd) of
~1 µM) from its l-enantiomer, for which no binding was
observed45. Aptamers can also function as the station-
ary phase in capillary electrochromatography46. The power
of these affinity applications is shown by a study that
generated an RNA affinity tag to allow the purification
of spliceosomes. Anti-tobramycin sequences were incor-
porated into pre-mRNA, which allowed large-scale puri-
fication of native spliceosomes through an interaction
with tobramycin immobilized on a resin47.
These same properties make aptamers ideal for use
in assays developed previously for antibodies, such as
Western blots or chromatin immunoprecipitation (ChIP)
assays48. Aptamers have distinct advantages over antibod-
ies in such assays as they usually have higher affinity and
specificity, giving much better signal-to-noise ratios,
and they can theoretically be raised against any protein,
including those which are poorly immunogenic or toxic.
They are usually smaller than antibodies allowing them
to bind to apatopes that are sterically inaccessible to
immuno globulins (see below for anti-HIV glycoprotein
120 (gp120) aptamers). They are also chemically modified
easily by routine processes.
Aptamers can be used to monitor the phosphor-
ylation state of proteins, giving information about the
temporal activity of proteins in signalling cascades and
biochemical pathways49. Using aptamers to alter the
activity of proteins in these pathways could be useful for
dissecting protein functions in vivo. However, it would
be of greater use if this effect were reversible so that the
protein could be activated and deactivated at will. Such
regulatable aptamers can be isolated by including a small
molecule effector that dissociates the aptamer from its
target protein at the elution step of the SELEX protocol.
The resulting aptamers bind and inhibit their targets but
dissociate from them when challenged with the same
effector, thereby restoring activity50. Another way to
regulate aptamers is by the Watson–Crick base-pairing
potential of complimentary RNA molecules (anti-sense
RNAs). The function of an aptamer selected against the
blood coagulation factor IXa (REF. 51) has been regulated
by addition of the anti-sense RNA, potentially providing
an alternative method for controlling coagulation in
patients who are intolerant of heparin. It also provides
a method for generating regulatable aptamer-based
therapeutic and diagnostic reagents.
An alternative method of aptamer regulation was
recently reported using ‘caged’, photolabile thymi-
dine residues in the well-characterized anti-thrombin
aptamer52,53. These modified residues were placed at
functionally important positions in the aptamer, com-
pletely inhibiting its binding. On UV irradiation the pro-
tecting groups were removed, restoring full activity and
demonstrating one route for precise spatial and temporal
regulation of aptamer activity.
Diagnostics and biosensors. The high affinity and
specificity of aptamers make them ideal diagnostic
reagents. Most diagnostic applications of aptamers rely
on ligand-induced conformational changes. These can
be detected by differential dye binding, fluorescence
quenching or fluorescence resonance energy transfer.
So called ‘aptamer beacons’ have many uses, which
range from detecting environmental contaminants to
monitoring carcinogen or drug levels in the blood54.
The description of modular aptameric sensors rep-
resents another step in the use of aptamers as biosen-
sors55. In these systems, a ‘recognition aptamer’ for the
ligand of interest is coupled to a ‘signalling aptamer’
by direct fusion of their nucleic-acid sequences. In
theory, the tandem aptamers could be incubated with
a sample of interest, allowing the recognition domain
to bind. The aptamers and any complexes they have
formed could then be washed with a dye solution that
binds to the signalling domain only when the ligand of
choice is bound, highlighting samples containing spe-
cific ligands. This simple system has a major advantage
in that the recognition domain does not require any
modifications that might adversely affect its structure
or function, allowing facile coupling of the many
ligand-binding and dye-binding aptamers already
characterized. In proof-of-principle experiments, the
anti-theophylline aptamer was coupled to the anti-
malachite-green aptamer. Binding of theophylline
resulted in conformational changes in the two-domain
aptamer, which allowed malachite-green binding. The
extension of this technique to a multiplexed array is
obvious, although this has yet to become commonplace
despite the fact that the use of aptamers in large scale,
diagnostic arrays was described as long ago as 1999
(REF. 56). However, with the development of automated
high-throughput aptamer isolation, modification and
screening, aptamer-based microarrays are now being
developed57, 58 (FIG. 3).
The recent development of quantum-dot aptamer
beacons could also help to establish a role for aptamers
in biosensors59. Quantum dots are novel fluorophores,
each having a distinct sharp emission profile, but they
can all be excited at the same wavelength. In quantum-dot
aptamer beacons, multiple copies of an aptamer are bound
to a single quantum dot. Each aptamer is base paired to
a complimentary strand carrying a quencher. The com-
plement is displaced on ligand binding, resulting in large
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cDNA microarray Aptamer microarray
Aptamer capture of protein
Polylysine coatingStreptavidin coating
A common three-dimensional
feature of RNA, in which bases
in a single-stranded loop base
pair with complementary bases
outside that loop. Pseudoknots
are commonly used
recognition and control
elements in vivo but often
stabilize selected aptamers.
increases in fluorescence emission. If different aptamers
are immobilized onto each quantum dot, multiple ligands
can be detected in a single assay. Such highly specific
and sensitive aptamer beacons have great potential as
early warning detection systems by binding cell-surface
apatopes specific for damaged or diseased cells.
An interesting development in the diagnostic applica-
tion of aptamers was recently reported in a simple assay
based on ligand interactions with aptamers bound to the
surface of gold nanoparticles60. Ligand binding induces a
conformational change in the aptamer leading to disas-
sembly of the aggregated nanoparticles. This disassembly
affects the absorbance properties of the nanoparticles,
resulting in a visible colour change. These studies could
be extended to develop aptamers used for the simple,
rapid detection of illegal drugs or substances associated
Therapeutics. Aptamers have enormous potential as
diagnostic reagents, which is only a stone’s throw away
from targeting aptamers for the treatment of disease.
Therapeutic agents, such as erythromycin and Tamiflu,
are traditionally small organic molecules that fit
snugly into clefts on the surface of their target macro-
molecule, forming an intricate network of stabilizing
interactions61,62. Aptamers can also fit into crevices on
macromolecules and can fold to form clefts into which
protruding parts of the target protein can bind. This
increases the potential number of contacts made with
the target, allowing aptamers to form tighter, more
specific interactions than smaller molecules.
Potential therapeutic targets can be divided into two
classes, intracellular targets, such as transcription fac-
tors, and extracellular targets, such as invading viruses.
Aptamers against extracellular targets can be adminis-
tered intravenously or subcutaneously. Pharmacokinetic
studies in humans confirm that RNAs delivered by
these routes are readily distributed throughout the
body and are easily taken up by cells63. The simplic-
ity of this approach is one of its main advantages. The
aptamers can be prepared in their stable functional state
and injected directly into the patient. However, RNA
degradation and clearance is inevitable, and repeated
administration is required until treatment is complete.
The delivery of aptamers to defined anatomical loca-
tions is already an approach in clinical use (see below)
and topical applications, for instance to prevent patho-
gens from interacting with their receptors on mucosal
surfaces, show potential.
Delivery of aptamers to intracellular targets has been
mostly by incorporation into liposome vesicles or by
expression from viral-based vector systems. A technique
using a fusigenic viral liposome vector to deliver DNA
aptamers to their target cells showed that DNA decoys
that sequestered the proliferogenic transcription factor
E2F led to a reduction in the abnormal vascular tissue
growth that is typically seen after angioplasty64. By con-
trast, other groups suggest that whereas conjugation of
carrier molecules to RNA might be required for uptake
by cells cultured in vitro, it is not necessary in vivo and
that “clinical formulations require only simple saline
Although these approaches show promise for deliv-
ery of aptamers to their intracellular targets, some
groups have begun to shift from studying the delivery
of aptamers to studying their expression in cells. Such
‘intramers’ are introduced by transfecting cells with a
retroviral vector encoding the aptamer. The transfected
cells then continuously produce the aptamer, in theory
providing life-long treatment. An example of this tech-
nique is the transient expression of a chimeric transcript
consisting of a human initiator tRNAMet sequence and
the anti-HIV reverse transcriptase pseudoknot aptamer
under the control of an RNA polymerase III promoter
in human 293 T cells. The chimeric RNA resulted in
>75% reduction in viral replication. Similar results were
seen with stably transfected Jurkat cells66. Obviously,
intramers do not rely on modified nucleotides for
stability. However, it has been shown that flanking the
aptamer sequence with stable stem-loop structures can
increase resistance to 3–5′ exonuclease attack67 , thereby
achieving a similar result.
The idea of using nucleic acids as therapeutic agents
is not a new one. In 1990, ‘RNA decoys’ with the same
sequence as the TAR RNA of HIV were shown to pre-
vent HIV replication in cells by sequestering all avail-
able Tat protein68. However, aptamer technology was
only used for the first time in clinical therapy 15 years
later, in early 2005. The Food and Drug Administration’s
approval of Eyetech/Pfizer’s aptamer (Macugen) for
the treatment of age-related macular degeneration is
a milestone in the applications of aptamer technology.
Figure 3 | Schematic showing the use of aptamers in array formats. The figure
compares the formats of complimentary DNA (cDNA) and aptamer microarrays.
Immobilization in the cDNA microarray is by a charge interaction between the DNA
and a polylysine-coated glass slide. In the aptamer microarray, the aptamers can
easily be biotinylated at one end allowing capture on a streptavidin-coated surface.
In the cDNA microarray each spot is hybridized to differentially labelled fluorescent
cDNAs prepared under the different test conditions and the result is analysed for the
relative amounts of each dye, which indicates the relative amount of cDNA in each
sample. A similar approach can be used with anti-protein aptamers if the proteins
are differentially labelled as shown. Figure reproduced with permission from REF 57
© (2005) Elsevier.
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Macugen is targeted against the angiogenic cytokine
vascular endothelial growth factor, and binding pre-
vents choroidal neovascularization16,69. This new class
of therapeutics is expected to be joined by inhibitory
aptamers against several other growth factors (includ-
ing platelet-derived growth factor and basic fibroblast
growth factor) for the treatment of cancers (for a review,
see REF. 70). There are also several groups developing
aptamers against amyloidogenic proteins such as the
Aβ-peptide associated with Alzheimer disease71, and
against abnormal proteins found in prion diseases72,73
such as scrapie and Creutzfeldt–Jakob disease.
Combating infectious agents. Aptamers also have the
potential to be used as anti-infectious agents. RNAs
can function as antibiotics if selected to inhibit a
crucial bacterial protein or to disrupt cell membrane
formation. Aptamers could also be used as a ‘targeting
system’, specifically binding and carrying an antibiotic
agent to the pathogen. An example of this approach
used an aptamer to deliver a low-affinity inhibitor to
Two active areas of interest are the use of aptamers
as inhibitors of the chronic viral infections HIV (for a
review, see REF. 75) and hepatitis C. There are several
key stages in the HIV life cycle that can be targeted by
aptamers, although so far inhibiting HIV replication
has been the main approach for treating patients. Most
recently available HIV treatments include nucleoside
analogues, which lack the 3′OH group and therefore
lead to premature termination of DNA elongation by
the HIV reverse transcriptase (RT). Unfortunately,
these analogues also inhibit normal cellular DNA
replication, resulting in severe side effects. In addition,
resistance can arise through single point mutations in
the RT active site. Several groups have isolated anti-RT
aptamers76,77 and one such pseudo knot aptamer binds
RT with low-nanomolar affinity. It has been shown
to cover ~2,600 Å2 of the RT surface, including the
DNA-binding region. It is thought that resistance to
such an inhibitor is unlikely to develop because of the
extensive protein alterations that would be required to
prevent aptamer binding.
Other anti-HIV drugs target the HIV protease.
Protease-deficient mutants cannot cleave the Gag–Pol
polyprotein to release functional viral proteins and are
therefore non-infectious. The absolute requirement
of HIV for functional protease makes it an ideal drug
target. Similar to RT inhibitors, however, a single point
mutation can make the protease resistant to current
drug therapies. Coupled with the fact that HIV protease
inhibitors also affect endogenous proteases leading to
severe side effects, it is clear that the search for more
effective treatments such as aptamer-based therapy must
HIV integrase catalyses the insertion of the nascent
dsDNA into the host genome. As the protease integrase
is crucial for viral replication but there is no human
equivalent of this enzyme, side effects from inhibitors
should be minimal. Several groups are focusing on
developing anti-integrase aptamers and one set of RNA
aptamers with a Kd of ~2 nM in vitro has been reported.
However their efficacy in cell-based assays has yet to be
The inhibition of viral invasion using aptamers
is another important area of HIV research. This has
advantages over the inhibition of viral replication as
the aptamers function extracellularly, obviating the
need to enter or be expressed in infected cells. Cell
invasion by HIV is a two-step process mediated by the
viral glycoproteins gp120 and gp41. Following an inter-
action between gp120 and CD4 receptors on the cell
surface, conformational changes allow gp41 to interact
with the chemokine receptors CCR5 or CXCR4. This
in turn induces a series of structural rearrangements in
the gp41 helical regions (HR) 1 and 2, resulting in the
formation of a ‘six-helix bundle’. This rearrangement
pulls the two membranes into close proximity allowing
fusion and therefore infection. The first FDA-approved
HIV-invasion inhibitor was a peptide corresponding
to HR2 (amino acids 127–162) of gp41. This peptide
binds to HR1 and inhibits the formation of the six-helix
bundle. Several groups have targeted aptamers against
the fusion process with the aim of inhibiting viral inva-
sion of the host cell. Anti-gp120 aptamers that bind
recombinant gp120 with a Kd of ~5–100 nM have been
reported and one of these, B4, inhibits HIV cell fusion
by up to 10,000-fold in cell culture79,80. Interestingly
these aptamers seem to bind to a cryptic site that is only
exposed when gp120 has bound to CD4 and is sterically
inaccessible to antibodies.
Several groups have recently reported aptamers tar-
geted against proteins of hepatitis C virus (HCV), the
causative agent of chronic hepatitis, liver cirrhosis and
hepatocellular carcinoma. The HCV non-structural pro-
tein NS3 is a crucial multifunctional component of HCV.
It has three known enzymatic activities: serine protease,
nucleoside triphosphatase and helicase activity. NS3 is
therefore an attractive target for in vitro selection, and
the isolation of domain-specific inhibitory aptamers has
recently been reported81–85. Another important HCV
protein target is the RNA-dependent RNA polymerase
NS5b (REF. 86). Aptamers that target NS5b have been
shown to inhibit RNA polymerase activity in vitro.
Aptamers have also been selected that function as inhibi-
tors of the highly conserved internal ribosome entry site
(IRES)87, and others directed against the apical loop of
IRES domain IIId have been shown to inhibit translation
of the viral genome both in vitro and in vivo88.
Although HIV and HCV are currently the main targets
for anti-viral aptamers, the isolation of aptamers against a
range of viral targets, such as human influenza virus and
cytomegalovirus has also been described89. Presumably,
similar approaches would allow the development of
aptamers that target avian influenza.
The results and developments described above show how
aptamer technology is rapidly maturing from a simple
research tool into a major technology with commercial
potential. Given that aptamers mimic and extend many
of the features of monoclonal antibody reagents, we must
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expect a similar development of commercial applications
over the next few years. In addition, the rapid expan-
sion in our understanding of the function and molecular
mechanisms of natural riboswitches are likely to spur
newer formulations of the basic aptamer concept. The
Ellington laboratory has recently isolated an aptamer
domain that will deliver molecular cargoes into cells
(A. Ellington, personal communication). The Harris
and Stonehouse laboratories have also selected aptam-
ers against HIV Nef and the foot and mouth disease
virus (FMDV) polymerase, respectively (M. Harris and
N. Stonehouse, personal communications). Although the
anti-Nef aptamers are only just being analysed, it is clear
that some of the anti-FMDV polymerase aptamers func-
tion as non-competitive inhibitors, binding adjacent to
the enzyme active site. In collaboration with the von Laer
group in Frankfurt, we have selected aptamers against
the HR2 region of HIV gp41 and have shown that the
resultant aptamer pools are inhibitory across many viral
strains (D.H.J.B. and P.G.S., unpublished observations).
It is therefore possible that a range of anti-viral aptamers
can be generated easily and that these might show syn-
ergistic activity, opening up new prospects for anti-viral
prophylaxis or therapy. It is clear that no one working
in the field of molecular microbiology can ignore the
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We would like to thank D. Burke, A. Ellington, M. Famulok
and W. James for helpful comments during the preparation
of this manuscript and for sharing unpublished or recent work
from their laboratories. We thank W. Horn for providing fig-
ure 2. Aptamer research in the P.G.S. laboratory is supported
by the UK Medical Research Council and the Biotechnology
and Biological Sciences Research Council, and by The
Wellcome Trust and The Leverhulme Trust.
Competing interests statement
The authors declare no competing financial interests.
The following terms in this article are linked online to:
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
E2F | gp41 | gp120 | Nef
Peter Stockley’s homepage: http://www.astbury.leeds.ac.uk
The Ellington Laboratory Aptamer Database: http://
Access to this links box is available online.
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