Review

Advances in SELEX and application of aptamers in the

central nervous system
Yan Yang
a
, Dongliang Yang
a
, Hermann J. Schluesener
b
, Zhiren Zhang
b,
*
a
Experimental Medical Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
b
Institute of Brain Research, University of Tuebingen, Calwer Street 3, D-72076 Tuebingen, Germany
Received 7 May 2007; received in revised form 12 June 2007; accepted 13 June 2007
Abstract
SELEX(Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly
specic ligands by repeated rounds of partition and amplication from a large combinatorial nucleic acid library. The products of the selection are
called aptamers, which are short single stranded DNA or RNA molecules, binding with high afnity, attributed to their specic three-dimensional
shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method
described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and
others. These new variants greatly shorten time of selection and improve aptamer afnity and specicity. Such aptamers have great potential as
detecting and/or diagnostic reagents. Furthermore, some aptamers specically inhibit biological functions of targeted proteins, and are considered
as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug
Administration of US for treating age-related macular degeneration. This review presents recent advances in the eld of SELEX with special
emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.
# 2007 Elsevier B.V. All rights reserved.
Keywords: SELEX; Aptamers; Central nervous system
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
2. Recent advances in the eld of SELEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
2.1. Capillary electrophoresis SELEX (CE-SELEX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
2.2. Tailored SELEX or primer-free SELEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
2.3. Toggle-SELEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
2.4. Expression cassette SELEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
2.5. Photo-SELEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
2.6. Automated SELEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
3. The expanding applications of aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
3.1. Analytical application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
3.2. Diagnostics and biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
3.3. Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
4. Aptamers in the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
4.1. Transmissible spongiform encephalopathies (TSE), or prion diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
4.2. Alzheimers disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
4.3. Myasthenia gravis and drug addicts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
4.4. Brain tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
4.5. Central regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
www.elsevier.com/locate/geneanabioeng
Biomolecular Engineering 24 (2007) 583592
* Corresponding author. Tel.: +49 7071 2984882; fax: +49 7071 294846.
E-mail address: zhangzhiren@yahoo.com (Z. Zhang).
1389-0344/$ see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bioeng.2007.06.003
5. Prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
1. Introduction
SELEX (Systematic Evolution of Ligands by Exponential
Enrichment) is a screening technique that involves the
progressive selection of highly specic ligands by repeated
rounds of partition and amplication from a large combina-
torial nucleic acid library. It was initially introduced by two
groups and based on general nucleic acid and protein
separation techniques (Tuerk and Gold, 1990; Ellington and
Szostak, 1990). It has proven to be an excellent tool for
selection of nucleotide polymers with high afnity for a
particular target froma randompool under specic conditions.
It involves three processes, namely: selection of ligand
sequences that bind to a target, partitioning of aptamers from
non-aptamers, and amplication of bound aptamers (Fig. 1). In
the primary step, a random single stranded nucleotide pool
must be designed and synthesized for conventional SELEX.
The length of the random sequence region is about 20100 nt.
During the selection process, the nucleic acid pools are treated
with the target molecule under appropriate buffer and
temperature conditions. After binding, partitioning of the
RNA/DNA aptamer-target complex from nonspecic mole-
cules can be achieved by various partitioning techniques and
only bound species are regenerated by enzymatic amplication
processes. These amplied molecules are used in the next
round of the selection process. Through repeated amplication
and several selection cycles, the nucleic acid molecules
binding to the target with high afnity and specicity are
enriched. Such combinatorial nucleic acid library can yield
ligands to many different molecules because of the ability of
single-stranded nucleic acid molecules (especially RNA
molecules) to fold into three-dimensional shapes with exible
structure that easily fold into pocket structure to bind with
target molecules.
The products of SELEX are called aptamers. Aptamers are
different fromantibodies, yet they mimic properties of antibodies
in a variety of molecular recognition formats (Jayasena, 1999).
Aptamers are identied through an in vitro process that does not
depend on animals, cells, or even in vivo conditions. As a result,
the properties of aptamers can be changed on demand. Because
animals or cells are not involved in aptamer identication, toxins,
as well as molecules that do not elicit good immune responses,
can also be used to generate high-afnity aptamers. Aptamers are
produced by chemical synthesis with extreme accuracy and
reproducibility. They are puried under denaturing conditions to
a very high degree of purity. Therefore, little or no batch-to-batch
variation is expected in aptamer production. Reporter molecules
such as uorescein and biotin can be attached to aptamers at
precise locations identied by the user. Functional groups that
allow subsequent derivatization of aptamers with other
molecules can also be attached during the chemical synthesis
of aptamers. After denaturation, functional aptamers could be
regenerated easily within minutes. They are stable in long-term
storage and can be transported at ambient temperature. The more
important is that aptamers can bind to a wider array of target
molecules including proteins, nucleic acids, peptides, amino
acids, organics, or even metal ions. The afnity and specicity of
aptamer-target binding complex are higher than that of antigen
antibody complex. These characteristics make aptamers a useful
tool in the diagnostic, analytical and therapeutic medical elds
(Bunka and Stockley, 2006).
Various improvement of the original SELEX method
described in 1990 has been reported during the recent decade,
such as capillary electrophoresis SELEX, Tailored-SELEX,
Fig. 1. Schematic of the conventional SELEX process. A random nucleotide pool is incubated with the target molecule; various partitioning techniques such as
membrane lter, afnity column, or titer plate are used to separate the RNA/DNA aptamer-target complex from nonspecic molecules; The bound molecules are
regenerated by amplication processes for the next selection cycle.
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 584
Toggle-SELEX, Photo-SELEX, and others. These new variants
greatly shorten time of selection and improve aptamer afnity
and specicity. This reviewpresents recent advances in the eld
of SELEX, with special emphasis on applications of aptamers
as analytical, diagnostic and therapeutic tools in the central
nervous system.
2. Recent advances in the eld of SELEX
2.1. Capillary electrophoresis SELEX (CE-SELEX)
Often in conventional SELEX, the target molecule has to be
attached to a stationary support by a linker molecule. The linker
eliminates a potential binding site on the target, preventing
sequences from interacting fully with the target. Another
important bias in conventional SELEX is the unfavorable
kinetics involved in the elution of high afnity sequences off
the column. Sequences that are strongly bound to the target are
difcult to wash off the column, resulting in a kinetic bias
against the best binders. Filtration techniques allow binding to
occur in free solution and thus eliminate linker bias. However,
because of the poor separation efciency of ltration
techniques, a large number of selection rounds are required.
Considerable improvements have, however, been made to
the selection step improving the efciency of selection, this is,
reducing the number of cycles or the time taken to isolate high-
afnity species. High-afnity DNA aptamers had been isolated
in a few rounds using capillary electrophoresis to separate free
protein or nucleic acids from complexed material (Mendonsa
and Bowser, 2004a,b, 2005). The process was named capillary
electrophoresis SELEX (CE-SELEX). CE-SELEX involves the
selection of binding species based on a mobility shift due to
complex formation with the target. As shown in Fig. 2, to
perform CE-SELEX, the nucleic acid library is incubated with
the target in a sample vial. The sample is injected into the
capillary and separated using free solution CE, regardless of
size or sequence. This is an advantage of CE-SELEX since all
unbound DNA migrates as a single band. Sequences bound to
the target travel through the capillary at a velocity different
from those not bound to the target, allowing binding sequences
to be isolated from non-binding sequences. It is a relatively
simple matter of collecting the two bands into separate
collection vials to separate binding from non-binding
sequences. The other steps of the process are similar to those
of conventional SELEX. PCR is used to amplify the sequences
that show afnity for the target. ssDNA is isolated and puried
following PCR to generate a new nucleotide pool for
subsequent rounds of CE selection. Advantages of CE-SELEX
include increased separation power, reduced nonspecic
binding, and ability to perform the selection in free solution.
As a result of these advantages, high afnity aptamers can be
obtained in 24 rounds of selection instead of the 812 typical
of conventional selections. Using CE-SELEX, the high-afnity
aptamers against IgE was selected with four selection rounds
(Mendonsa and Bowser, 2004a,b). For this selection, the
authors used a poly(vinylalcohol)-coated 40.2 cm long capil-
lary with an inner diameter of 50 mm for separation. The
sample was applied to the capillary at 5 psi for 5 s and
monitored under UV detection at 254 nm. After the nonspecic
species had migrated out, the CE fractions containing specic
DNA sequences were collected. The collected DNA sequences
were amplied by PCRand used for the next round of selection.
Using a similar strategy, an ssDNA sequence was selected
against a small peptide, neuropeptide Y (Mendonsa and
Bowser, 2005). This selection procedure proved that CE-
SELEX was also suitable for smaller targets.
Recently, equilibrium capillary electrophoresis of equili-
brium mixtures has been introduced to select an aptamer
against MutS protein with three rounds of selection processes
only (Drabovich et al., 2005). Further, a non-SELEX selection
process for aptamers was also introduced; involving repeated
partitioning steps without amplication (Berezovski et al.,
2006). Here non-equilibrium capillary electrophoresis of
equilibrium mixtures for partitioning was used, and it took
1 h to complete the selection processes, which increased the
selected molecule afnity by four orders of magnitude. In this
study, with h-Ras protein as the target, aptamers were selected
from a DNA library of 39 random sequences labeled with 6-
carboxyuorescein at the 5
0
end. The main advantage of this
method was that the selection processes could be completed
within 1 h instead of several days or weeks.
2.2. Tailored SELEX or primer-free SELEX
The primary nucleic acid pool of the SELEX process usually
comprise 6090 nts, with 3040 nts long randomized regions
plus xed primer sites of 1525 nts on each side. Therefore, the
identied lead oligonucleotides often need rational and
experimental truncation before they can be further tested in
biological system. But to date only short RNAs or DNAs with
less than 60 nt are efciently produced chemically at reasonable
Fig. 2. Capillary electrophoresis SELEX scheme. The nucleic acid library is
incubated with the target protein. The mixtures are injected into the capillary
and separated using free solution CE. Binding sequences are isolated from
nonbinding sequences based on a mobility shift induced by complex formation
with the target protein. Bound sequences are collected and PCR amplied,
preparing a new pool for further rounds of selections.
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 585
costs. Furthermore, the xed regions, although facilitating PCR
amplication and in vitro transcription, often give an impact on
the binding of aptamer-target complexes. The xed regions are
also possibly annealing with the randomized region that give an
impact tothe implement of selection. Therefore, time-consuming
experimental truncation processes that do not always lead to the
desired results need to be carried out before aptamers are
available. Variations of the SELEX protocol have allowed
isolation of truncated aptamers with desired properties. Tailored-
SELEX, for example, involves ligation and cleavage of primer
sites before and after amplication, allowing the isolation of
shorter aptamer sequences that are more readily synthesized
chemically and the direct and rapid isolation of target binding
RNA sequences (Vater et al., 2003). Tailored-SELEX process
uses four and six xed nucleotides at the 5
0
and 3
0
endof a primer-
less RNAlibrary, respectively. The primer binding sequences are
ligated to the library using three double-stranded molecules
(adapters) and removed within the amplication procedure
without the need of purication steps. Tailored-SELEXhas been
successfully applied to identify high afnity oligonucleotides to
calcitonin gene-related peptide (Vater et al., 2003). The Tailored-
SELEX approach is based on a ligation strategy for the primer-
binding site to an RNA library carrying a small number of xed
nucleotides on both sides of the randomized region. Compared to
a traditional selection, only two additional reaction steps are
needed. Recently, a RNAlibrary design have been developed that
can be used in two different constitutions: as a (conventional)
full-length library with primer binding sites or as a short library
without primer binding sites (Jarosch et al., 2006). The library
carries seven complementary (xed) nucleotides that clamp the
randomized region and constrains the oligonucleotides into a
partly double-stranded structure. This design already minimizes
the risk that the primer binding sites become part of the target-
bindingmotif. However, the use of primer bindingsites forminga
double-stranded structure does not guarantee their truncation
without reduction or loss of binding to the target. Therefore,
moreover, the sequence arrangement of the dual library also
allows carrying out the selection step with the shorter library. In
this case the anking primer binding sites are removed before the
selection step and added back subsequently. The structural
constraints of the RNA library and the specic use of the short
library within the selection step facilitate the direct identication
of short aptamer sequences without the need of time-consuming
or unsuccessful truncation experiments. However, in order to
limit potential losses of valuable target binding sequences due to
additional enzymatic steps, in early selection rounds only the
full-length library is employed. After a solid basis of target-
binding (full-length) sequences is achieved (here after nine
selection rounds), the selection protocol is switched to the shorter
library. This switch avoids enrichment of sequences which
functions are potentially dependent on the existence of primer
binding sites.
Genomic SELEX is an in vitro selection-amplication
method proposed for identication of biologically important
nucleic acidprotein interactions. It is a method for studying the
network of nucleic acidprotein interactions within any
organism. Like SELEX of randomized sequence nucleic acids,
genomic SELEX consists of repeated rounds of binding a
library of nucleic acids to the target protein, separating the
bound nucleic acids from the unbound ones and amplifying the
bound ones for the next round. The starting libraries in genomic
SELEX, unlike the randomized sequence SELEX, are derived
from the genome of the organism of interest. Genomic SELEX
is conceptually related to other high-throughput methods of
functional analysis of the genomes, such as the one-hybrid,
two-hybrid and three-hybrid systems. It can be used to
construct nucleic acidprotein linkage map, similar to protein
protein linkage maps. A problem with genomic SELEX is that
the primer-annealing sequences of the library may base pair
with the central genomic fragments to form structures that are
selected as sites for target binding. A method named primer-
free genomic SELEX has been developed to circumvent these
articial effects (Wen and Gray, 2004). That is, primer-
annealing sequences are removed from the genomic library
before selection with the target protein and are then regenerated
to allowamplication of the selected genomic fragments. Akey
step in the regeneration of primer-annealing sequences is to
employ thermal cycles of hybridization-extension, using the
sequences from unselected pools as templates. Primer-free
genomic SELEX offers a method to identify potentially
biologically important nucleic acid sequences for target
molecules while simultaneously reducing articial effects.
2.3. Toggle-SELEX
The intricate nature of the proteinaptamer interaction
results in the production of aptamers which are highly specic
for the target protein. For example, aptamers are capable of
discriminating between isoforms of protein kinase C that share
a high degree of identity. But some times the species cross-
reactivity is desired in the pre-clinical evaluation of a molecule
in animal models. Some potential therapeutic molecules that
demonstrate excellent efcacy in vitro might fail to enter
clinical trials due to lack of compelling efcacy in animal
experiment. For therapeutic molecules generated against
human protein targets, decreased afnity for the homologous
proteins in appropriate animal models may be one reason for
lack of in vivo efcacy. Therefore, in vitro selection strategies
that facilitate the generation of therapeutic agents that are cross-
reactive between species have been developed (White et al.,
2001). The selection process was named as Toggle-SELEX and
allowed isolation of aptamers with a broader range of
specicities by selecting against related targets in alternating
cycles. Alternation of the target between homologous proteins
of different species ensures that the products of selection will
bind to both proteins, most likely to domains conserved
between the two proteins. Thereby RNA ligands that bind both
human and porcine thrombin by toggling the protein target
between human and porcine thrombin during alternating rounds
of selection during alternating rounds have been selected
(White et al., 2001). Such cross-reactive aptamers inhibit both
porcine and human thrombin activity, as might be expected
from ligands made to bind evolutionary conserved regions of a
protein. The toggle strategy is a simple measure that promotes
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 586
cross-reactivity and may be generalizable to related proteins of
the same species. Toggle-SELEX should facilitate the isolation
of ligands with needed properties for gene therapy and other
therapeutic and diagnostic applications.
2.4. Expression cassette SELEX
Aptamers are fascinating new reagents to be used as
diagnostic tools as well as therapeutics to battle a variety of
diseases. However, the main obstacle remains their delivery.
This is especially problematic when the target is localized
inside the cell. The development of aptamers for gene therapy
has been complicated by the fact that expression of RNA
aptamers in the context of anking sequences can inhibit the
ability of an aptamer to fold into its functional conformation. A
retroviral vector for intracellular expression of a chimeric RNA
consisting of the human initiator tRNA
Met
/aptamer sequence
that binds to the human immunodeciency virus (HIV) type I
reverse transcriptase in human 293T cells has been described
(Chaloin et al., 2002). Binding studies indicate that the
pseudoknot motif is properly folded in the sequence context of
the tRNA
Met
and the extra RNA parts at the two terminal ends
are not interfering with binding to its target. Transient
intracellular expression of the chimeric aptamer shows
inhibition of HIV particle release by more than 75% when
cells were co-transfected with proviral HIV-1 DNA. Further, it
has been reported that insertion of the E2F aptamer into a tRNA
expression cassette resulted in the production of high levels of
chimeric tRNA that retained high afnity binding to E2F1 and
which could be expressed at high levels in mammalian cells
(Martell et al., 2002). Moreover, these chimeric tRNA-E2F
aptamers are functional and inhibit E2F-mediated transactiva-
tion by up to 80% in human 293 cells. The application of this
aptamers by gene transfer techniques represents a very
promising and innovative approach to treat infections with
viruses. Expression cassette SELEX should greatly facilitate
the use of aptamers for a variety of gene therapy applications.
2.5. Photo-SELEX
Photo-SELEX involves UV-induced cross-linking of apta-
mers containing light-sensitive nucleotides to their target,
greatly increasing binding afnity. It envisioned that a
combinatorial methodology whose selection criterion is the
ability to form a photo-induced covalent bond to the target
molecule might yield aptamers with substantially greater
sensitivity and specicity than those evolved by methodologies
utilizing the afnity selection criterion. In the PhotoSELEX
variation of SELEX, a modied nucleotide activated by
absorption of light is incorporated in place of a native base in
either RNA- or in ssDNA-randomized oligonucleotide
libraries. One such modied nucleotide whose photochemistry
is particularly well suited for this purpose is 5-bromo-2
0
-
deoxyuridine (BrdU). The 5-bromouracil chromophore absorbs
UV light in the 310 nm range where native chromophores of
nucleic acids and proteins do not absorb or absorb very weakly.
The resulting excited singlet state intersystem crosses to the
lowest triplet state which specically cross-links with aromatic
and sulfur-bearing amino acid residues of a protein target in
suitable proximity. Using this method, with a random 61-mer
oligonucleotide library in which 5-bromo-2
0
-deoxyuridine
replaced thymidine, aptamers against the recombinant human
basic broblast growth factor155 (bFGF155) were selected
(Golden et al., 2000) and two aptamers showed exquisite
photocross-link-yield with bFGF155 and detected bFGF155
with exceptional sensitivity and simultaneously, readily
distinguished bFGF155 from consanguineous heparin-binding
growth factors. BrdU-photoaptamers, are rapidly emerging as
specic protein capture reagents in protein microarray
technologies. A mathematical model for the kinetic analysis
of photoaptamerprotein photocross-linking reactions has been
presented (Koch et al., 2004). The model is based on specic
aptamer/protein binding followed by laser excitation that can
lead to either covalent cross-linking of the photoaptamer and
protein in the complex or irreversible photodamage to the
aptamer. The models are used to characterize the photocross-
linking between three photoaptamers and their cognate protein
targets. Data for cross-linking reaction yields as a function of
both laser energy dose and target protein concentration are
analyzed for afnity constants and cross-link reaction rates.
The binding dissociation constants derived from the cross-
linking data are in good accord with independent measure-
ments. This work provides a useful set of metrics that will allow
further renement of photoaptamer properties.
2.6. Automated SELEX
In the past few years, various attempts at automating of
SELEX have been made (Cox and Ellington, 2001; Cox et al.,
2002; Eulberg et al., 2005). Automated systems provide for
high exibility and versatility in terms of choice of buffers,
reagents and stringency conditions during selections. Auto-
mated systems can handle multiple targets efciently since they
can be processed in parallel. Moreover, selection cycles can be
conducted without any direct intervention steps. In 2001,
automated workstations were used to select anti-protein
aptamers, and demonstrated the automated selection of anti-
lysosme that functions as an efcient inhibitor of cell lysis (Cox
and Ellington, 2001). Further improvements resulted into an
automated aptamer selection procedure coupled to a protein
translational system to select aptamers against protein targets
generated by in vitro transcription and translation from
individual genes (Cox et al., 2002). For specic immobilization
they also used in vitro biotinylation. To prove the applicability
of this method, aptamers against translated human U1A, a
component of the nuclear spliceosome, were selected. Based on
this high throughput method, aptamers against the translation
products of the target genes within an average bacterial genome
can be found within a period of months. Eulbergs team also
selected the RNA aptamers against the mirror image
conguration (D-peptide) of substance P, the so called
Spiegelmer, using an automated system (Eulberg et al.,
2005). Very recently, a microudic SELEX prototype was
described as being an automated microuidic, microline-based
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 587
assembly with Labview-controlled actuatable valves and a PCR
machine to verify the selection and synthesis of antilysozyme
aptamer (Hybarger et al., 2006). According to their view, this
system is relatively inexpensive and useful for the continued
morphing of macro ! meso ! microfabricated structure
and applicable for more rapid selection. The rapid selection of
aptamer ligands by this automated system and its options for
controlling experimental settings should provide many oppor-
tunities in several elds.
3. The expanding applications of aptamers
Due to their easy and quick preparation, cost-effective-
ness, small size and versatility, aptamers have become useful
tools for the validation of intracellular and extracellular
targets. A continuously growing number of nucleic acid
aptamers are used as research tools to study specic protein
functions and interactions (Huang et al., 2004; Cao and Tan,
2005; Hwang and Nishikawa, 2006). Moreover, aptamers are
used to study regulatory circuits and molecular mechanisms
of disease processes (Gopinath et al., 2006). Aptamers can
readily be adopted for analytical or diagnostic applications
(German et al., 1998; Wang et al., 2004; Vivekananda and
Kiel, 2006; Centi et al., 2007). In vivo experiments
demonstrate that they generally exhibit low toxicity and
immunogenicity characteristics, which make them suitable
for therapeutical use as well (Que-Gewirth and Sullenger,
2007).
3.1. Analytical application
The versatility of aptamers is reected by the fact that
there are few areas of research to which aptamers cannot be
applied. One of the most obvious uses for these highly
specic, high-afnity, reusable molecules is as an afnity
purication medium. An advantage of aptamers over most
other solid media for protein purication is that the pure
protein can be obtained in fewer steps, owing to the ability of
aptamers to discriminate between closely related ligands.
Furthermore, aptamers can be selected to bind to the natural
form of the protein, eliminating modications with tags (such
as 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 is to use an immobilized anti-
selectin aptamer to purify a selectin-receptor globulin
expressed in Chinese hamster ovary cells (Romig et al.,
1999). Aptamers can be used to monitor the phosphorylation
state of proteins as well, giving information about the
temporal activity of proteins in signaling cascades and
biochemical pathways (Seiwert et al., 2000). Further, using
aptamers to alter the activity of proteins in these pathways
could be useful for dissecting protein functions in vivo. An
alternative method of aptamer regulation was recently
reported using caged, photolabile thymidine residues in
the well-characterized anti-thrombin aptamer (Heckel and
Mayer, 2005; Mayer et al., 2005). These modied residues
were placed at functionally important positions in the
aptamer, completely inhibiting its binding. On UV irradia-
tion, the protecting groups were removed, restoring full
activity and demonstrating one route for precise spatial and
temporal regulation of aptamer activity.
3.2. Diagnostics and biosensors
The high afnity and specicity of aptamers make them
ideal diagnostic reagents. Many diagnostic applications of
aptamers rely on ligand-induced conformational changes.
These can be detected by differential dye binding,
uorescence quenching or uorescence resonance energy
transfer. So called aptamer beacons have many applica-
tions, which range from detecting environmental contami-
nants to monitoring carcinogen or drug levels in the blood
(Brockstedt et al., 2003). The description of modular
aptameric sensors represents another step in the use of
aptamers as biosensors (Stojanovic and Kolpashchikov,
2004). 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 signaling
domain only when the ligand of choice is bound, highlighting
samples containing specic ligands. This simple system has a
major advantage in that the recognition domain does not
require any modications that might adversely affect its
structure or function, allowing facile coupling of the
many ligand-binding and dye-binding aptamers already
characterized.
3.3. Therapeutics
Aptamers have enormous potential as diagnostic reagents,
which is only a stones throw away from targeting aptamers for
the treatment of disease. Therapeutic agents, such as
erythromycin and Tamiu, are small organic molecules that
t snugly into clefts on the surface of their target macro-
molecule, forming an intricate network of stabilizing interac-
tions (Tu et al., 2005; Varghese et al., 1995). Aptamers can also
t 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 specic interactions
than smaller molecules. Aptamers 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, specically binding and carrying an
antibiotic agent to the pathogen. An example of this approach
used an aptamer to deliver a low-afnity inhibitor to its target
(Charlton et al., 1997). The active area of interest is the use of
aptamers as inhibitors of the chronic viral infections HIV (Held
et al., 2006).
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 588
4. Aptamers in the central nervous system
4.1. Transmissible spongiform encephalopathies (TSE), or
prion diseases
Transmissible spongiform encephalopathies (TSEs) or prion
diseases are neurodegenerative disorders that include kuru and
CreutzfeldtJakob disease in humans as well as mad cow
and other diseases in animals. A critical initiating event in the
pathophysiology of TSEs is the conversion of cellular prion
protein (PrP
C
) to an aberrant isoform called PrP
SC
(the scrapie
form). This isoform forms high-molecular-weight protease K
(PK) resistant aggregates that accumulate in the central nervous
system of affected individuals (Hussein and Al-Mufarrej,
2004).
The biochemical processes involved in prion propagation,
prion processing, and prion protein aggregation, are largely
unknown. Aptamers for infectious prions have been isolated in
order to investigate conformational aspects of prion pathogen-
esis (Weiss et al., 1997). SELEX technique was used to select
RNA aptamers that bind to the prion protein PrP
C
. RNA
oligomers that recognized PrP
C
contained four sets of three-
guanine base repeats that formed a G quartet scaffold. The
RNA binding site mapped to the NH2 terminus of PrP
C
(residues 2352). This work suggests the possibility of using
SELEX technique to obtain aptamers that would bind to the
infectious isoform PrP
SC
of this protein and could, therefore, be
used as a diagnostic tool for spongiform encephalopathies
(Proske et al., 2002a,b; Rhie et al., 2003). Isolated 2
0
-
uoropyrimidine-substituted RNA aptamers that bind selec-
tively to disease-associated-bsheet-rich forms of the prion
protein, PrP
SC
were shown to reduce the proportion of PrP
SC
in
neuroblastoma cells and in a physiological cell-free conversion
system, respectively. An internal site for biotinylation of a
minimized, synthetic aptamer had been identied and is used in
the detection of abnormal forms of PrP in vitro (Sayer et al.,
2004).
DNA aptamers that bind specically to recombinant human
PrP
C
and mammalian PrP
C
with varying afnities were selected
and can be applied to biological samples for PrP
C
enrichment
and as diagnostic tools in double ligand assay systems
(Takemura et al., 2006). The preferred RNA sequences that
bind to the ovine recombinant PrP were identied using
SELEX. A minimal sequence necessary for binding of selected
aptamer to PrP presents a striking similarity with one
previously described PrP aptamer of comparable afnity.
Further, two lysine clusters contained in the N-terminal part of
PrP are essential to nucleic-acid binding (Mercey et al., 2006).
RNA aptamers against the mouse prion protein (mPrP) was
selected and the 2
0
-uoro-pyrimidine modications for RNase
resistance did not abolish its binding activity. In addition,
protein-deletion-mutant analysis and competition-binding
analysis using heparin showed that the aptamer appears to
have binding sites located between amino acids 23108 (Sekiya
et al., 2006). These aptamers could be potentially drugs for
prionprotein associated diseases, but this would require a
mean for transport across the blood brain barrier.
4.2. Alzheimers disease
The beta-amyloid peptide (Abeta) is a major component of
the Alzheimers disease (AD)-associated senile plaques and is
generated by sequential cleavage of the beta-amyloid precursor
protein (APP) by beta-secretase and gamma-secretase. Since
BACE1 initiates Abeta generation it represents a valuable target
to interfere with Abeta production and treatment of AD. While
the enzymatic activity of BACE1 resides in the extracellular
domain, the protein also contains a short cytoplasmic tail (B1-
CT). This domain serves as a binding site for at least two
proteins, the copper chaperone for superoxide dismutase-1
(CCS), and the Golgi-localized, gamma-ear-containing, ADP
ribosylation factor-binding (GGA1) protein, and contains a
single phosphorylation site. However, the precise role of the
B1-CT for the overall biological function of this protein is
largely unknown. Functional studies focusing on the activity of
this domain would strongly benet from the availability of
domain-specic inhibitors. Rentmeister As team described the
isolation and characterization of RNA aptamers that selectively
target the B1-CT. They showed that these RNAs bind to
authentic BACE1 and the binding site is restricted to the
membrane-proximal half of the C terminus. Aptamer-binding
specically interferes with the recruitment of CCS, but still
permits GGA1 association and casein kinase-dependent
phosphorylation, consistent with selective binding site target-
ing within this short peptide. Because phosphorylation and
GGA1 binding to B1-CTregulate BACE1 transport, these RNA
inhibitors could be applied to investigate B1-CT activity
without affecting the subcellular localization of BACE1
(Rentmeister et al., 2006).
4.3. Myasthenia gravis and drug addicts
The nicotinic acetylcholine receptor (AChR) controls signal
transmission between cells in the nervous system. Myasthenia
gravis is a neuromuscular disorder characterized by muscular
weakness and fatigue resulting from antibody-mediated auto-
immune response to AchR (Drachman, 1994). A 2
0
-amino-
modied aptamer bound to Mab198, a monoclonal antibody that
recognizes the major immunogenic epitope onhumanAchR, was
isolated (Lee and Sullenger, 1997). This aptamer protected
TE671 cells from antigenic down-modulation in a dose-
dependent manner and also protected AChRfromautoantibodies
found in patients with myasthenia gravis (Lee and Sullenger,
1997). A subsequent selection with 2
0
-uoropyrimidine-mod-
ied RNA library isolated an improved Mab128 aptamer that is
evenmore effective at protectingAChRfromthe monoclonal and
patient autoantibodies (Seo and Lee, 2000).
Abused drugs such as cocaine inhibit the nicotinic
acetylcholine receptor. Transient kinetic investigations indicate
that inhibitors decrease the channel-opening equilibrium
constant. Can compounds be found that compete with inhibitors
for their binding site but do not change the channel-opening
equilibrium? SELEX methodology was applied in Torpedo
californica electroplax membranes to select RNA aptamers that
can displace inhibitors from the receptor. Two classes of
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 589
aptamers were selected and both are displaced by phencyclidine
and cocaine from their binding site on the AChR. Class I RNA
aptamers are potent inhibitors of AChR activity in BC3H1
muscle cells, as determined by using the whole-cell current-
recording technique. Class II RNA aptamers, while competing
with AChR inhibitors, do not affect receptor activity in this
assay, suggesting that such compounds or derivatives may be
useful for alleviating the toxicity experienced by millions of
addicts (Ulrich et al., 1998). However, these ligands are
relatively unstable. To increase the stability of these RNA
aptamers, chemical modication was used. Two classes of 2
0
-
uoro-modied RNA aptamers were obtained. One class binds
with higher afnity to the cocaine-binding site on the closed-
channel form and, as predicted by the mechanism, inhibits the
receptor. The second class binds with equal or higher afnity to
the cocaine-binding site on the open-channel form and does not
inhibit the receptor, and does alleviate cocaine and MK-801
inhibition of the nAChR. The high stability of these 2
0
-uoro-
RNAs expands the utility of these ligands (Cui et al., 2004).
4.4. Brain tumor
Tenascin-C (TN-C) is a large, extracellular matrix glyco-
protein that is overexpressed during tissue remodeling such as
wound healing, atherosclerosis, psoriasis, and tumor growth
(Erickson and Bourdon, 1989). Anti-TN-C aptamers that are
related in sequence have been isolated using both puried TN-C
and TN-C-expressing glioblastoma cells (Hicke et al., 2001;
Daniels et al., 2003). A size-minimized and nuclease-stabilized
aptamer, TTA1, binds to the brinogen-like domain of TN-C.
This 13 kDa aptamer is intermediate in size between peptides
and single chain antibody fragments, both of which are superior
to antibodies for tumour targeting because of their smaller size.
TTA1 denes a new class of ligands that are intended for
targeted delivery of radioisotopes or chemical agents to
diseased tissues.
Targeting large transmembrane molecules, including recep-
tor tyrosine kinases (RTK), is a major pharmacological
challenge. The RET (rearranged during transfection) RTK is
physiologically stimulated by any member of the glial cell line-
derived neurotrophic factor (GDNF) family. Using RET-
expressing cells as targets in a modied SELEX procedure,
nuclease-resistant aptamers that recognize the human RET
were obtained. One of these aptamers blocked RET-dependent
intracellular signaling pathways by interfering with receptor
dimerization when the latter was induced by the physiological
ligand or by an activating mutation. This strategy is generally
applicable to transmembrane receptors and opens the way to
targeting other members of this class of proteins that are of
major biomedical importance (Cerchia et al., 2005).
In several neuroblastoma cell lines, retinoic acid (RA)-
induced differentiation is coupled to increased expression of
RET. To study the specic role of RET in RA-induced
differentiation of SK-N-BE(2) cell line, Laura Cerchia team
using a 2
0
-uoro-RNA aptamer and a truncated RET protein as
specic inhibitors of RET, nd that RA-induced differentiation
is mediated by a positive autocrine loop that sustains RET
downstream signaling and depends on glial cell-derived
neurotrophic factor expression and release. This report shows
that in SK-N-BE(2) cells, stimulation of RET is a major
upstream mechanism needed to mediate RA-induced differ-
entiation. These results provide important insights on the
molecular mechanism of RA action, which might be relevant
for the development of biologically based therapeutic strategies
(Cerchia et al., 2006).
4.5. Central regulation
The neuropeptide nociceptin/orphanin FQ (N/OFQ), which
is distributed throughout the central nervous system, is an
endogenous agonist of the G protein-coupled opioid receptor-
like 1 (ORL1) receptor and found both in the central nervous
system and peripheral tissues. It has been shown that N/OFQ
plays a prominent role in the regulation of biological functions
such as pain, anxiety, motor impairment, and others, which
makes N/OFQ and ORL1 potential therapeutic targets (Calo
et al., 2000). Although a series of agonists and antagonists of
the ORL1 receptor as well as anti-N/OFQantibodies exist, none
of these show the specicity and potency necessary for their
development as therapeutics untill now (Calo et al., 2002).
Using a mirror image in vitro selection approach, biostable
RNA aptamers (Spiegelmers) specicly to N/OFQ was isolated
(Faulhammer et al., 2004). Spiegelmers are L-enantiomeric
oligonucleotide ligands that display high afnity and specicity
to their targets and high resistance to enzymatic degradation
compared to D-oligonucleotides. This Spiegelmers were shown
to antagonize binding of N/OFQ to the ORL1 receptor in a
binding-competition assay. Furthermore, this Spiegelmers
antagonized the N/OFQ-induced GTPgS incorporation into
cell membranes in a CHO-K1 cell line expressing the human
ORL1 receptor. In oocytes from Xenopus laevis, this
Spiegelmers showed an antagonistic effect to the N/OFQ-
ORL1 receptor system that was functionally coupled with G-
protein-regulated inwardly rectifying K
+
channels. Therefore,
this Spiegelmers not only specically bind to N/OFQ but also
interferes the physiological functions of N/OFQ, indicating a
high potential as an alternative therapeutic substance class for
the N/OFQ-ORL1-receptor system of this Spiegelmers.
Neuropeptide Y (NPY) that is a 36-amino acid neuropeptide
is widely distributed in the central and peripheral nervous
system and modulates a variety of physiological processes such
as the central regulation of food intake, vasoconstriction,
memory retention, and regulation of circadian rhythm. NPY
transmits its activity by at least three receptor subtypes (Y1, Y2,
and Y5), which all belong to the large family of G-protein-
coupled receptors (GPCR) (Loftus et al., 2000; Bischoff and
Michel, 1999). To understand how NPY selectively activates a
particular receptor pathway and characterize the interaction
between the peptide and its receptors, a nuclease-resistant RNA
aptamer directed against NPY had been isolated (Proske et al.,
2002a,b). This aptamer bound well to Y2 receptor-specic
NPY mutants but not to pancreatic polypeptide, a highly
homologous Y4 receptor-specic peptide of the gut, the Y5
receptor-selective agonist NPYand the Y1/Y5 receptor-binding
Y. Yang et al. / Biomolecular Engineering 24 (2007) 583592 590
peptide NPY. Accordingly, the structure of the aptamer was
highly similar to the NPYepitope which was recognized by the
Y2 receptor. Further, this aptamer mimics the binding of NPY
to the Y2 receptor more closely than to the Y1 and Y5
receptors. The study suggests that NPY binding by the aptamer
is strikingly similar to that of the Y2 receptor. This in turn
justies the assumption that NPY may exist in different
biologically active conformations in vivo including the
possibility that the Y2 receptor-specic NPY conformation
differs from the conformation that is required for the activation
of the Y1 or Y5 receptors.
G-protein-coupled receptors (GPCRs) are integral mem-
brane proteins involved in signal transduction and constitute
major drug targets for therapy. Aptamers could represent a
valuable tool to probe the role of such receptors in normal and
pathological tissue and for co-crystallization with receptors for
structure determination by X-ray crystallography. As an
example of this strategy, aptamers directed against the rat
neurotensin receptor NTS-1 were isolated (Daniels et al.,
2002). One of the aptamers was characterized in detail and
shown to bind to both the rat receptor and the human receptor
with nanomolar afnity. The aptamer was also demonstrated to
interact with rat neurotensin receptors expressed in membrane
preparations and in intact cells.
5. Prospective
During the past two decades, aptamer technology has
developed from a simple research tool to an important
biotechnology which might be an alternative to monoclonal
antibody technology in the future due to its power and
exibility of in vitro selection. Considerable advances to
improve the efciency of selection and to increase the afnity,
specicity, biostability and bioaviability of aptamer have paved
the road for practical/commercial applications of aptamer. With
the understanding of molecular mechanisms underlying central
nervous system diseases, aptamer technology represents an
exciting class of compounds for the development of new
therapeutic and diagnostic agents.
Acknowledgements
We thank Prof. Guo, Peixuan (Cancer Research Center,
Purdue University, USA) for nucleic acid library and valuable
comments and suggestions. This work is supported by BMBF
(03134298) and NSFC grants (30571646).
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