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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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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