This document describes an experiment to develop a method for selecting aptamers against protein targets generated through in vitro transcription and translation of genes. Specifically, they attempt to select aptamers against the human U1A protein, a component of the nuclear spliceosome, where the U1A protein was produced through in vitro transcription and translation of its gene, and was also biotinylated to allow for immobilization during aptamer selection. The results showed that the selected aptamer sequences closely mimicked the natural RNA binding sequences and structures of U1A, demonstrating the potential of this method for high-throughput aptamer generation against proteomes.

1. Automated selection of aptamers against protein
targets translated in vitro: from gene to aptamer
J. Colin Cox1
, Andrew Hayhurst2
, Jay Hesselberth3
, Travis S. Bayer1
, George Georgiou1,2
and Andrew D. Ellington1,3,*
1
Institute for Cellular and Molecular Biology, 2
Department of Chemical Engineering and 3
Department of Chemistry
and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
Received May 31, 2002; Revised July 18, 2002: Accepted August 8, 2002
ABSTRACT
Reagents for proteome research must of necessity
be generated by high throughput methods. Apta-
mers are potentially useful as reagents to identify
and quantitate individual proteins, yet are currently
produced for the most part by manual selection pro-
cedures. We have developed automated selection
methods, but must still individually purify protein
targets. Therefore, we have attempted to select apta-
mers against protein targets generated by in vitro
transcription and translation of individual genes. In
order to speci®cally immobilize the protein targets
for selection, they are also biotinylated in vitro. As a
proof of this method, we have selected aptamers
against translated human U1A, a component of the
nuclear spliceosome. Selected sequences demon-
strated exquisite mimicry of natural binding
sequences and structures. These results not only
reveal a potential path to the high throughput gener-
ation of aptamers, but also yield insights into the
incredible speci®city of the U1A protein for its
natural RNA ligands.
INTRODUCTION
The development of in vitro selection methods has potentiated
the identi®cation of nucleic acid-binding species (aptamers)
and catalysts (aptazymes) that are proving increasingly useful
in therapeutic and diagnostic applications (1±3). However, as
information continues to accrue about the sequences of
organismal genomes and the composition of organismal
proteomes, it will be necessary to increase the throughput of
aptamer selection methods in order to either quickly identify
aptamers against novel targets or to generate aptamers against
entire proteomes or metabolomes.
To this end, we have previously developed methods for the
automated selection of aptamers (4±6). We initially estab-
lished a rudimentary set of chemistries and robotic manipu-
lations that facilitated the basic execution of nucleic acid
selection and ampli®cation (5). The original automated
system was used for the selection of aptamers against
isolated oligonucleotide or protein targets. For example, the
non-nucleic acid-binding protein lysozyme was immobilized
on streptavidin beads and anti-lysozyme aptamers that had
reasonable af®nities (Kd » 31 nM) for the cognate protein and
that inhibited enzymatic function were selected (6). The same
procedures were applied in short order to a variety of other
proteins, including CYT-18 (a tyrosyl-tRNA synthetase from
the mitochondria of the fungus Neurospora crassa), MEK1 (a
human MAP kinase kinase) and Rho (a transcriptional
termination protein from the archaebacterium Thermotoga
maritima). Each of the aptamers generated by automated
selection procedures have demonstrated high speci®city and
af®nity for their targets (Kd values in the pico- to mid-
nanomolar range) (4). The current automated selection system
can yield aptamers against virtually any compatible target
within 3 days.
However, automated selection methods are still too low
throughput to contemplate the acquisition of aptamers against
all of the protein targets in a proteome. One of the primary
limitations on the method is that target proteins must be
individually acquired, most frequently by puri®cation from an
overproducer strain. To avoid this bottleneck, we have now
attempted to generate aptamers against protein targets that
have been directly transcribed and translated on the robotic
workstation. Blackwell and Weintraub previously demon-
strated that manual selections with double-stranded DNA
libraries could be carried out against protein targets translated
in vitro (7).
An initial selection was carried out against translated U1A
protein, a component of the human spliceosome. The wild-
type sequences that U1A binds to have been characterized in
great detail (8±10) and the anti-U1A aptamers that were
generated by automated selection methods mimic the wild-
type sequences in every important detail. These results suggest
that automated selection methods may soon prove capable of
deciphering speci®c interactions between multiple different
nucleic acid-binding proteins and cellular RNA sequences.
MATERIALS AND METHODS
Oligonucleotides, genes and plasmids
The N-terminal RNA-binding domain of U1A was synthe-
sized from 15 oligonucleotides using a PCR-based assembly
method (11). The sequence of U1A was derived from
GenBank accession no. M60784, modi®ed to include the
*To whom correspondence should be addressed. Tel: +1 512 471 6445; Fax: +1 512 471 7014; Email: andy.ellington@mail.utexas.edu
ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 20 e108

2. same mutations (Y31H and Q36R) that were present in the
protein crystallized by Oubridge et al. (12). The assembled
gene was sub-cloned into pET28a, yielding a gene encoding a
fusion protein with an N-terminal 6Q histidine tag. This was
placed into an expression vector to create pJH-hisU1A. The
sequence of the ®nal construct was veri®ed.
The assembled U1A gene fragment was used to generate a
template for in vitro transcription and translation (TnT)
reactions. Speci®cally, `splice-overlap extension' (SOE) PCR
(13) was used to attach sequences necessary to direct high
level expression and quantitative biotinylation of U1A during
in vitro coupled transcription±translation reactions (Fig. 1).
The primers used during these experiments are described in
Table 1.
The 5¢ region for SOE PCR is ~150 bp in length. The T7
gene 10 promoter and translation initiation region (TIR) are
included to enhance transcript stability and translation (14,15).
Protein synthesis is then initiated with a 24 amino acid biotin
acceptor peptide sequence (`biotag', MAGGLNDIFEAQKIE-
WHEDTGGSS) (16). A megaprimer containing both the TIR
and the biotag was generated using primer AHX10, primer
AHX47 and the Escherichia coli biotinylation and expression
plasmid pDW363 (17). The 3¢ region is ~1050 bp in length.
The birA gene is followed by the major transcription
Figure 1. Scheme for in vitro transcription, translation and biotinylation. Splice-overlap extension was used to assemble 5¢ and 3¢ fragments onto genes of
interest. The 5¢ fragment contained a T7 promoter and biotin protein ligase (BPL) recognition sequence (`biotag'). The 3¢ fragment contained the gene for
BPL (birA) and a T7 terminator. Transcription generated a dicistronic mRNA that when translated yielded a `biotagged' gene of interest and functional BPL.
Free biotin was covalently attached to the gene of interest by BPL and the tagged protein was puri®ed from the reaction by capture with strepavidin.
e108 Nucleic Acids Research, 2002, Vol. 30 No. 20 PAGE 2 OF 14

3. terminator signal for T7 RNA polymerase to enhance
transcript stability (14). A megaprimer containing birA and
the transcription terminator was generated using primer
AHX33, primer AHX29 and pDW363. The assembled U1A
gene was PCR ampli®ed with primers (U1A.biotag and
U1A.t0) that added overlap regions complementary to both
megaprimers. The 3¢ assembly junction creates an opal
termination codon followed by a functional RBS for birA
(17). SOE assembly was carried out with the two `rescue'
primers AHX31 and AHX30.
Initial in vitro transcription, translation and
biotinylation experiments
The maltose-binding protein (MBP) gene with the N-terminal
biotin acceptor peptide from pDW363 (17) was PCR ampli®ed
using primers AHX10 and AHX32. The ®nal product
contained a suitable ribosome-binding site for birA after the
mbp termination codon. Standard PCR and SOE were
performed using YieldAce DNA polymerase (Stratagene, La
Jolla, CA), which outperformed other polymerases in terms of
yield, especially with SOE. Primers were gel puri®ed to ensure
ef®cient SOE. In order to determine whether endogenous
biotin protein ligase (BPL) was suf®cient to biotinylate protein
targets, the mbp gene was separately merged with two
different birA variants (18). The ®rst construct was generated
using primers AHX29 and AHX55 and encoded a short, ®ve
amino acid fragment of the birA gene (MKDNT) followed by
the T7 major terminator. The second construct was generated
using primers AHX33 and AHX29 and encoded a full-length
birA gene. The ®nal SOE products were ampli®ed using
primers AHX30 and AHX31. DNA was puri®ed using a
QIAquick PCR puri®cation kit (Qiagen, Valencia, CA).
For the in vitro transcription and translation reaction, ~1 mg
of template was introduced into 50 ml T7 PROTEINscript-
PRO reactions (Ambion, Austin, TX) supplemented with
10 mM D-biotin. Reactions were incubated at 37°C for 60 min.
A control reaction without template was carried out simul-
taneously. Reactions were centrifuged (~13 000 g, 15 min,
4°C). The supernatants were removed and designated `soluble
fraction' while the pellets were resuspended in 50 ml of water
and designated `insoluble fraction'. Samples (10 ml) were
combined with Laemmli sample buffer (10 ml), boiled and
loaded onto 10% Laemmli gels (19). Rainbow molecular
weight markers (Amersham Pharmacia Biotech, Piscataway,
NJ) were employed as standards.
Following semi-dry transfer to Immobilon P PVDF
(Millipore, Bedford, MA), the membranes were blocked
overnight with ~25 ml of PBS containing 2% BSA (w/v). One
membrane was probed with a 1:10 000 dilution (v/v) of rabbit
anti-MBP serum (New England Biolabs, Beverly, MA)
followed by a 1:30 000 dilution of anti-rabbit IgG±
horseradish peroxidase (HRP) (Bio-Rad, Hercules, CA) to
detect MBP. The other membrane was probed with a 1:100 000
dilution of avidin-conjugated HRP (Sigma-Aldrich, St Louis,
MO) to detect biotinylated products. Sera probes were
incubated with blots for 30±60 min. Blots were washed in
100 ml of PBS + 0.1% Tween-20, four times for 10 min each
time. Blots were further washed with 2 Q 100 ml of PBS for
10 min each. Development was for a few seconds using a
SuperSignal West Pico chemiluminescence kit (Pierce,
Rockford, IL) and signals were captured on X-ray ®lm.
Having established that co-translation of BPL was neces-
sary to ensure ef®cient biotinylation of the MBP target, we
then applied the methodology to three other genes: chlor-
amphenicol acetyltransferase (cat), neomycin phosphotrans-
ferase II (neo) and b-lactamase (amp). The cat gene was
ampli®ed using primers AHX51 and AHX52 with plasmid
pHELP1 (available from the authors, derived from
pACYC184) as template (20). The neo gene was ampli®ed
using primers AHX49 and AHX50 with plasmid pIMS102
(available from the authors, derived from pNEO) as template.
The amp gene was ampli®ed using AHX53 and AHX54 with
Table 1. Primers listed in Materials and Methods
Priming sequences are shown 5¢®3¢.
PAGE 3 OF 14 Nucleic Acids Research, 2002, Vol. 30 No. 20 e108

4. plasmid pUC19 as template. SOE PCR was used to fuse each
of the genes to the T7 promoter, biotag, birA and terminator.
The dicistronic templates were puri®ed and employed in
in vitro transcription, translation and biotinylation reactions as
above.
Protein targets were assayed for biotinylation. The soluble
portions of reactions were applied to P6 desalting chromatog-
raphy columns (Bio-Rad) to remove free biotin and then
combined with 50 ml of glycerol to ensure precipitation-free
storage at ±20°C. A high-binding ELISA plate (Corning, New
York, NY) was coated overnight at 4°C with 1 mg/ml
Neutravidin (100 ml; Pierce) in 50 mM sodium bicarbonate
buffer, pH 9.6. The Neutravidin-coated plate and an uncoated
control plate were blocked overnight in ~350 ml of PBS with
2% BSA (block). The TnT reactions in glycerol (10 ml) were
serially diluted by factors of two in block and distributed onto
both Neutravidin and control plates (100 ml ®nal volumes).
After 1 h, plates were washed three times with ~350 ml of PBS
+ 0.1% Tween-20 and then twice with plain PBS. Primary
antisera speci®c for the gene products were applied in 100 ml
of block for 1 h. Anti-cat, anti-neo and anti-amp antisera
(Eppendorf±5 prime, Boulder, CO) were used at 1:1000
dilutions in 100 ml of PBS; anti-mbp antisera (New England
Biolabs) was used at a 1:10 000 dilution in 100 ml of
PBS. Following washing, anti-rabbit IgG±HRP (Bio-Rad
Laboratories) was applied at a 1:3000 dilution in 100 ml for
1 h. Following washing, the plates were developed for 10 min
with 100 ml of o-phenylenediamine (Sigma-Aldrich) at
0.4 mg/ml. o-Phenylenediamine was originally dissolved in
50 mM phosphate citrate buffer pH 5.0, with 4 ml of 30% H2O2
added per 10 ml. Color development was stopped with 50 ml of
2±3 M sulfuric acid and the OD was measured at 492 nm on an
ELISA plate reader (Bio-Tek, Winooski, VT). The blank plate
was subtracted from the experimental plate to correct for
background binding.
In vitro transcription and translation of U1A
The U1A±birA construct was transcribed and translated in vitro
using the RTS 100 E.coli HY kit (Roche Diagnostics, Basel,
Switzerland). In order to generate suf®cient protein for the
entire course of an automated selection experiment a quad-
ruple sized reaction (200 ml) was carried out starting with 2 mg
of DNA template. Biotin (14 mM ®nal concentration) was also
added at the start of the combined TnT reaction. The reaction
was incubated at 30°C for 6 h, followed by incubation at 4°C
for ~4 h. Unincorporated biotin was removed by passing the
entire reaction though a Bio-Spin 6 chromatography column
(Bio-Rad). The puri®ed, biotinylated protein sample was
incubated with 2.4 mg of strepavidin-coated magnetic
Dynabeads (Dynal, Oslo, Norway) in the presence of 1Q
selection binding buffer (1Q SBB = 20 mM Tris pH 7.5,
100 mM KOAc, 5 mM MgCl2) for 15 min. Beads were
thoroughly washed ®ve times in 500 ml of selection buffer to
remove any non-biotinylated protein.
Protein expression and biotinylation were veri®ed by
electroblotting and western blot analysis using strepavidin-
conjugated alkaline phosphatase as a reporter for biotinylated
proteins (Fig. 2). Aliquots of the TnT reaction (5 ml) were
placed in 1 ml of b-mercaptoethanol (b-ME), 10 ml of water
and 4 ml of 5Q SDS±PAGE loading dye (250 mM Tris±HCl
pH 6.8, 50% glycerol, 2.5% SDS, 142 mM b-ME, 0.05%
bromophenol blue) and heat-denatured by boiling for 5 min.
Half of the denatured sample (10 ml) was loaded onto a 12%
acrylamide (29:1 acrylamide:bisacrylamide) denaturing
protein gel on a MiniPROTEAN 3 gel rig (Bio-Rad). The
gel was run until the bromophenol blue dye reached the
bottom (~30 min at 200 V). The gel was electroblotted onto
Trans-Blot nitrocellulose (Bio-Rad) in a Mini Trans-Blot
Electrophoretic Transfer Cell (Bio-Rad) in electroblotting
buffer (10 mM NaHCO3, 3 mM NaCO3, 30% methanol,
pH 9.9) for 90 min at 350 mA. The blot was blocked with 1Q
Tris-buffered saline and Tween-20 (TBST) for 30 min at room
temperature with gentle agitation. Next, the blot was incu-
bated with 3 ml of alkaline phosphatase-conjugated strepavidin
(Promega, Madison, WI) in fresh 1Q TBST buffer (15 ml) for
30 min with mild agitation. Unbound alkaline phosphatase
was removed with three washes with 1Q TBST (15 ml, 5 min
each). Finally, the blot was brie¯y rinsed with deionized water
and placed in 15 ml of Western Blue Stabilized Substrate for
alkaline phosphatase (Promega) for 1±5 min.
In vitro selection
Automated in vitro selection was carried out as previously
described (4,6). In short, a RNA pool that contained 30
randomized positions (N30) (4,6,21) was used as a starting
point for selection. The RNA was incubated in 1Q SBB with
biotinylated protein immobilized on 200 mg of streptavidin
beads (suf®cient to bind 4±8 Q 1012 biotinylated antibody
molecules). RNA±protein complexes were ®ltered from free
RNA and weakly bound species were removed by washing
with 4 Q 250 ml of 1Q SBB. Bound RNA molecules were
eluted in water at 97°C for 3 min. RNA for additional rounds
of selection was generated by reverse transcription, PCR and
in vitro transcription (using primers 41.30 and 24.30). All of
the reactions were carried out sequentially on a Beckman-
Coulter (Fullerton, CA) Biomek 2000 automated laboratory
workstation that had been specially modi®ed and programmed
to interface with a PCR machine (MJ Research, Waltham,
MA) and a ®ltration plate device (Millipore).
Figure 2. Detection of translated, biotinylated U1A. Expression of biotinyl-
ated U1A was assayed by western blot analysis. Biotinylated protein size
standards are in the ®rst lane. The second lane shows a no template control,
while the third and fourth lanes show controls with gene products that were
not biotinylated. The ®fth lane contains an aliquot from an in vitro tran-
scription and translation reaction with the biotagged U1A gene. The arrow
indicates the translated, biotinylated U1A gene product. The ~20 kDa band
seen in all control and experimental lanes is thought to be biotin carboxy-
carrier protein (BCCP), the single biotinylated protein in E.coli.
e108 Nucleic Acids Research, 2002, Vol. 30 No. 20 PAGE 4 OF 14

5. Eighteen rounds of selection were performed against
biotinylated U1A. In the ®rst 12 rounds of selection 20 cycles
of PCR were carried out, while in the last six rounds this
number was decreased to 16 cycles to prevent overampli®ca-
tion of the selected pool; these parameters had been empiric-
ally determined during previous automated selection
experiments (6). In the ®rst 12 rounds of selection the wash
buffer was 1Q SBB. In the last six rounds of selection the
stringency of the selection was increased by progressively
increasing the monovalent salt concentration, as detailed in
Table 2.
The progress of selection was monitored every six rounds
(6, 12 and 18) by placing [a-32P]-radiolabeled ribo- and
deoxyribonucleotides in the ampli®cation reactions and
resolving ampli®cation products by gel electrophoresis. The
automated protocol included a provision to archive aliquots of
the reverse transcription (10 ml, 10% of the total reaction) and
in vitro transcription (10 ml, 10% of the total reaction)
reactions. After the automated protocol had run its course, the
aliquots (in standard stop dye) were run on 8% acrylamide±
7 M urea (19:1 acrylamide:bisacrylamide) denaturing gels.
Decade RNA Markers (Ambion) were used as size standards.
The gels were visualized using a PhosphorImager SI
(Amersham Pharmacia Biotech). In addition, single point
binding assays were carried out with equimolar concentrations
of protein and RNA samples (50 nM), as described below (see
also Fig. 5).
High throughput sequencing
Aliquots (1 ml of a 50 ml archive) of RT±PCR reactions from a
given round of selection were further ampli®ed and then
ligated into a thymidine-overhang vector (TA Cloning Kit;
Invitrogen). Templates for sequencing reactions were gener-
ated from individual colonies via 50 ml colony PCR reactions
(22) with standard M13 sequencing primers ¯anking the
insertion site of the thymidine-overhang vector (primers M13
forward and M13 reverse). The PCR products were puri®ed
away from primers, salt and enzyme using a MultiScreen96-
PCR clean-up plate (Millipore). Aliquots of the colony PCR
reactions (5 ml) were developed on a 4% agarose gel to verify
the insertion of aptamers into vectors.
Cycle sequencing reactions were performed using a CEQ
DTCS Quick Start Kit (Beckman-Coulter) and the vendor's
modi®ed M13 sequencing primer (primer ±47 seq). Reaction
assembly and cycling conditions were performed largely as
described in the vendor's instructions. Approximately
100 fmol of puri®ed colony PCR products were used as
templates and reactions were performed with half of the
master mix concentration recommended in the instructions in
order to minimize reagent use (4 ml of master mix in a 20 ml
sequencing reaction, rather than 8 ml). Unincorporated dye
was removed by size exclusion chromatography, as described
in Beckman-Coulter Technical Application Information
Bulletin T-1874A (http://www.beckman.com/Literature/
BioResearch/T-1874A.pdf). Brie¯y, 45 ml of dry Sephadex
G50 (Amersham-Pharmacia Biotech) was placed into each
well of a MultiScreen HV plate using a MultiScreen 45 ml
Column Leader (Millipore). The Sephadex chromatography
resin was allowed to hydrate in 300 ml of water for 3 h at room
temperature. After incubation, the resin was packed by
centrifugation for 5 min at 1100 g. The columns were rinsed
once with 150 ml of water and packed again at the same speed
for the same time. The 20 ml sequencing reactions were loaded
onto the tops of the columns using a multichannel micro-
pipettor and spun again for 5 min at 1100 g. Puri®ed samples
were recovered from a CEQ Sample microplate (Beckman-
Coulter) that had been placed below the chromatography
plates before centrifugation. Recovered samples were dried
under vacuum at room temperature. Pellets were resuspended
in 40 ml of deionized formamide and developed on a CEQ
2000XL eight channel capillary DNA sequencer (Beckman-
Coulter). Aptamer secondary structures were predicted using
RNAstructure 3.6 by Mathews et al. (23).
Cellular expression of U1A
The plasmid pJH-hisU1A was transformed into BL21 cells
(Stratagene). A 1 ml starter culture grown from a single
plasmid was used to inoculate 50 ml of fresh LB. The culture
was grown to an OD600 of ~0.6 at 37°C and protein expression
was induced by the addition of IPTG to a ®nal concentration of
840 mM. The induced culture was grown for an additional 3 h
at 37°C. Cells were pelleted at 5000 g and lysed in B-PER
(Pierce), 10 U DNase (Invitrogen) and 10 mM MgCl2 in a total
volume of 5 ml. After incubation at room temperature for
15 min, cellular debris was pelleted at 13 000 g. The
supernatant was further puri®ed by nickel-chelation chroma-
tography. IMAC resin (2 ml; Amersham-Pharmacia Biotech)
was equilibrated with 4 ml of water, followed by 4 ml of
charging buffer (50 mM NiCl2). The column was equilibrated
with 10 ml of protein binding buffer (PBB; 1Q PBB = 50 mM
Tris pH 7.5, 100 mM NaCl, 5 mM imidazole). Clari®ed
supernatant (~15 ml) was loaded and the column was washed
with 4 ml of 1Q PBB and 15 ml of wash buffer (50 mM Tris
pH 7.5, 100 mM NaCl, 50 mM imidazole). The U1A protein
was eluted by the addition of 3 ml of elution buffer (50 mM
Tris pH 7.5, 100 mM NaCl, 500 mM imidazole); 500 ml
fractions were collected for gel analysis. Fractions containing
signi®cant amounts of U1A were pooled and dialyzed in
Table 2. Conditions for the
automated selection of anti-U1A
aptamers
See Materials and Methods for details.
The binding ability of the pool was
assayed at 0, 6, 12 and 18 rounds.
PAGE 5 OF 14 Nucleic Acids Research, 2002, Vol. 30 No. 20 e108

6. 50 mM Tris pH 7.5, 100 mM NaCl with four buffer exchanges
of 500 ml every 3 h at 4°C in order to remove imidazole from
the preparation. Finally, the absorbance at 280 nm was used to
determine protein concentration (extinction coef®cient =
5442 M±1 cm±1).
Binding constants
Plasmids containing individual aptamers were used to gener-
ate transcription templates via PCR. Transcription reactions
were carried out with an AmpliScribe T7 RNA transcription
kit (Epicentre, Madison, WI) according to the manufacturer's
instructions, except that incubation was at 42°C rather than
37°C. Aptamers were puri®ed on denaturing polyacrylamide
gels (24), dephosphorylated and radiolabeled with [g-32P]ATP
(25). Radiolabeled RNA was extracted with phenol:chloro-
form (1:1) and unincorporated nucleotides were removed
using size exclusion spin columns (Princeton Separations,
Adelphia, NJ).
Nitrocellulose ®lter binding assays were employed to
determine the dissociation constants of aptamer±protein
complexes (25). A standard protocol was automated using
the Biomek 2000 automated laboratory workstation and a
modi®ed Minifold I ®ltration manifold (Schleicher & Schuell,
Keene, NH). RNA samples in 1Q SSB were thermally
equilibrated at 25°C for 30 min. The RNA concentration for
binding reactions was kept constant at a ®nal concentration of
200 pM while the concentration of U1A ranged from 1 mM
down to 17 pM (11 different concentrations). Equal volumes
(60 ml) of RNA and U1A were incubated together for 30 min at
room temperature in 1Q SBB. The binding reactions (100 ml)
were ®ltered through a sandwich of Protran pure nitrocellulose
(Schleicher & Schuell) and Hybond N+ nylon transfer
membrane (Amersham Pharmacia Biotech) that had been
assembled in the modi®ed Minifold I vacuum manifold. The
®lters were washed three times with 125 ml of 1Q SBB. The
amount of radiolabeled RNA captured from a reaction onto the
nitrocellulose membrane was quanititated using a Phosphor-
Imager SI (Amersham Pharmacia Biotech). The log of U1A
concentration was plotted against the amount of RNA bound.
Data were ®tted to the equation y = (a´b)/(b + x) + C, where C
is the fraction of RNA bound to the nitrocellulose at zero
protein concentration, b is the maximum fraction bound, x is
the fraction of RNA bound to U1A and a is the dissociation
constant for the RNA±protein complex. Assays were per-
formed in triplicate and standard deviations were calculated.
The 21 nt synthetic RNA (AAUCCAUUGCACUCCGGA-
UUU) previously employed in structural studies of the U1A
protein was used as a positive control (12).
RESULTS AND DISCUSSION
From gene to biotinylated protein
While we have previously automated the in vitro selection of
aptamers that target proteins (4±6), our methods are currently
limited to puri®ed proteins. Ultimately, the need to purify
protein targets individually would drastically reduce the speed
with which aptamers could be generated against proteomes
and would therefore reduce the utility of aptamers as reagents
for proteome analysis. Therefore, we have sought to increase
selection throughput by generating protein targets via in vitro
transcription and translation. Moreover, in order to manipulate
protein targets during automated selection, we have attempted
to introduce a biotin tag during the in vitro synthesis
procedure.
A number of kits were available for the in vitro transcription
and translation of genes. Ultimately, we found that the Roche
RTS 100 E.coli HY kit worked well with the automated
selection procedures we had previously established. In order to
biotinylate translated proteins, templates were modi®ed
(Fig. 1) so that translated proteins would contain an N-ter-
minal peptide tag (MAGGLNDIFEAQKIEWHEDTGGSS)
that was an ef®cient substrate for BPL, the product of the birA
gene (26±28). The e-amino group of the single lysine residue
in the BPL recognition peptide becomes covalently linked to
biotin (29). Biotin ligase can either be co-translated or added
as a separate reagent.
Existing BPL present in E.coli S30 extracts proved
insuf®cient to ef®ciently biotinylate target proteins.
Therefore, the MBP gene (malE) was expressed in tandem
with a downstream birA cistron. Following gel separation, an
anti-MBP antibody was used to con®rm that roughly equal
amounts of MBP were synthesized. However, when
avidin±HRP was used as a probe it was apparent that only
the dicistronic mbp±birA template directed ef®cient biotinyl-
ation. The band at ~20 kDa that stains intensely with
avidin±HRP is E.coli biotin carboxyl-carrier protein
(BCCP), which is present in the S30 extracts used for in vitro
translation (Fig. 2). MBP and three other biotinylated protein
targets were immobilized in Neutravidin-coated ELISA wells
and detected with cognate sera (Fig. 3). These results indicated
that in vitro translated and biotinylated proteins could fold into
native structures that present epitopes similar to those found
in vivo. The amounts of in vitro translation and biotinylation
reactions necessary to saturate microwells for selection
experiments proved to be relatively small (1% of reaction
volume), indicating that this procedure should be useful for
multiplex formats, such as the acquisition of aptamers against
multiple proteins in an organismal proteome.
We have previously carried out selections against chemical-
ly biotinylated target proteins (4,6). Unfortunately, chemical
biotinylation generally generates a population of molecules
with differing amounts of biotinylation at different conjuga-
tion sites, typically a-amino groups on a protein. Thus,
multiple different epitopes are presented during selection. A
biotinylation tag obviates this problem and a relatively
homogeneous set of epitopes should be present during the
selection. Additionally, chemical biotinylation may block an
active or allosteric site of the protein, while speci®c
biotinylation at the N-terminus is less likely to interfere with
function.
In vitro selection of aptamers that bind to translated,
biotinylated U1A
As in our previous automated selection experiments, biotinyl-
ated protein targets are loaded onto streptavidin beads and
incubated with RNA libraries. Bound RNA molecules are
sieved from unbound by ®ltration; the use of beads facilitates
robotic manipulation. The beads are directly transferred to a
thermal cycler and RNA is prepared for the next round of
selection by a combination of reverse transcription, PCR and
in vitro transcription. The entire selection procedure can be
e108 Nucleic Acids Research, 2002, Vol. 30 No. 20 PAGE 6 OF 14

7. iteratively carried out using a Beckman-Coulter Biomek 2000
automated laboratory workstation that has been specially
modi®ed and programmed to interface with a thermal cycler.
The selection robot can carry out six cycles of selection per
day; a typical selection is done in 2±3 days. This represents an
increase in speed of at least an order of magnitude relative to
manual selection methods, and up to eight targets can be
handled in parallel by the Biomek 2000. Overall, these
methods allow us to go from a gene sequence to high af®nity
binding reagents for the protein product of that gene in under
1 week.
As an initial target, we attempted to select aptamers against
a RNA-binding domain of the U1A protein. U1A is a member
of the U1 snRNP, a component of the nuclear spliceosome that
participates in processing of pre-mRNA splicing (30). The ®rst
96±100 amino acids of U1A are responsible for binding
hairpin II of U1 snRNA in the splicing complex (31). Its
N-terminal portion, containing one of the two RNP domains of
U1A, has been shown to maintain the binding characteristics
and sequence speci®city of the full-length protein (9,10). Most
importantly, U1A has previously been targeted by in vitro
selection experiments (8) and thus we could be assured that it
would likely generate aptamers.
As the selection progressed, the DNA and RNA pools
produced at each round were examined by denaturing gel
electrophoresis (Fig. 4). The lengths of the DNA and RNA
molecules were remarkably uniform, especially given that the
number of cycles and incubation times used for DNA and
RNA ampli®cation were pre-set. The consistent sizes of
ampli®cation products are in part a result of our prior
optimization of PCR for automated selection procedures
(4±6). The stringency of the selection was increased by
increasing the salt concentration from 100 mM during the ®rst
12 rounds (2 days) of selection to 300 mM at round 13. It is
interesting to note that the amount of RNA that is ampli®ed
following this round seems to drop, just as though the increase
in stringency reduced the amount of pool that survives the
selection. A further increase in stringency was introduced at
round 15 by increasing the salt concentration to 600 mM.
Filter binding assays were employed to detect signi®cant
changes in the af®nities of selected pools for U1A (Fig. 5).
The initial pool showed signi®cant binding to U1A, likely due
to electrostatic interactions with the highly charged target
protein (25,32) (+7.2 at pH 7). Binding assays were therefore
also carried out at higher salt concentrations (1 M) to reduce
this non-speci®c background binding signal. Six rounds of
automated selection yielded no signi®cant increase in apparent
af®nity, but a further six yielded an appreciable increase in
signal (1.7-fold increase in binding signal even in the presence
of high salt). The ®nal six rounds, carried out under
increasingly stringent conditions, yielded additional improve-
ments in pool binding (3.1-fold increase in binding signal even
in the presence of high salt). All binding assays were
performed using a his-tagged U1A isolated from E.coli, rather
than the biotin-tagged U1A generated by in vitro translation
and biotinylation. The fact that binding did not appear to be
dependent upon the type of tag present suggested that binding
was speci®c for the RNP. It is interesting that no aptamers
were isolated against BCCP, given that this protein was also
always present as a target. It is likely that RNP is a better target
for selection than BCCP. When `mock' selections were
carried out without the gene for U1A, aptamers against BCCP
were isolated (data not shown).
Anti-U1A aptamers resemble natural binding sequences
The natural RNA ligands of the U1A RNP have been well
characterized. Hairpin II of U1 snRNA is a short stem
topped by a G:C base pair and a loop with the sequence
Figure 3. Detection of in vitro expressed and biotinylated products. Four constructs were investigated for their ability to bind to neutravidin-coated microwells
after in vitro expression and biotinylation: b-lactamase (amp), chloramphenicol acetyltransferase (cat), aminoglycoside phosphotransferase (neo) and maltose-
binding protein (mbp). Puri®ed, translated and biotinylated products were serially diluted and probed with their respective antisera and again with anti-rabbit
IgG±HRP. Signal intensity is measured after incubation of the bound complexes with o-phenylenediamine at 492 nm.
PAGE 7 OF 14 Nucleic Acids Research, 2002, Vol. 30 No. 20 e108

8. 5¢-AUUGCACUCC (Fig. 6A). The bold heptamer sequence
has been shown to be critical for binding to U1A; for instance,
G9 makes hydrophobic contacts with Arg52 and hydrogen
bonding interactions with Arg52, Leu49, Asn15 and Lys50 of
U1A (33,34). The other six bases form similar hydrophobic,
hydrogen bonding and stacking interactions with other peptide
backbone and side chain positions in the U1A protein (12,34).
The last three bases (UCC) do not physically interact with the
protein and act primarily as a spacer to close the loop
(8,12,35,36). In addition, the U1A protein interacts in a similar
manner with the 3¢-UTR of its own mRNA (37,38),
autoregulating its own expression by inhibiting polyadeny-
lation and cleavage (12,39,40) (Fig. 6B). However, in this
instance the canonical heptamer sequence is presented as an
internal loop (Box II), rather than a hairpin loop. Following
binding of one U1A monomer to the Box II internal loop, a
second U1A monomer is recruited to bind to an adjacent
internal loop containing a variant of the canonical heptamer,
AUUGUAC (Box I).
Aptamers from rounds 6, 12 and 18 were cloned and
sequenced (Fig. 7). Aptamer sequences that contained the
canonical heptamer were seen at the sixth round of selection:
8% (1 of 12 clones) contained a perfect match to the heptamer,
while 42% (5 of 12 clones) had imperfect matches to this
sequence (Fig. 7A). After another six rounds of selection the
sequence diversity of the pool was further reduced and most
individuals (79%, 11 of 14 clones) possessed the heptamer or
near perfect matches (14%, 2 of 14 clones; Fig. 7B). Further
selection with increasing stringency yielded a pool where 87%
of sequences (26 of 30 clones) contained the heptamer and the
remaining 13% (4 of 30 clones) had near perfect matches
(Fig. 7C). No sequences lacked perfect or near perfect matches
in the ®nal pool.
Based on similar sequences found outside of the conserved
heptamer, anti-U1A aptamers could be clustered into two
sequence families. Family 1 makes up 33% of the ®nal pool,
and can be further subdivided into ®ve clonal sub-families, all
with the consensus sequence UGRACAUUGCACUACG.
Family 2 comprises 23% of the ®nal population of the pool
and is largely clonal.
Predictions of the secondary structures of individual
aptamers are consistent with the hypothesis that Family 1
mimics the wild-type U1 snRNA hairpin II, while Family 2 is
instead structurally similar to the 3¢-UTR of U1A mRNA
(Fig. 6C and D). It should be noted that aptamers that are not in
Family 1 or Family 2 may still generally resemble the wild-
type ligands. For example, many of the aptamers listed as
`other perfect matches' (Fig. 7C) can readily be folded to form
hairpin II-like structures.
Interestingly, all residues that have previously been shown
by structural or functional analyses to be important for
hydrophobic, stacking or hydrogen bonding interactions were
conserved in the selected aptamers. Conversely, residues that
are known to not be important for interactions with U1A were
not conserved. The degree to which features of natural ligands
have been recapitulated by in vitro selection is remarkable. A
C:G base pair closes the loop in the wild-type U1 snRNA
hairpin and has been shown to be crucial for binding an
arginine residue in U1A. Disruption of this base pair and this
interaction eliminates U1 snRNA binding to U1A (9,41). All
Figure 4. Nucleic acid products during automated U1A selection. Radioactive reaction products from RT±PCR and transcription reactions during automated
selection were resolved on denaturing polyacrylamide gels. RNA (80 nt) and DNA (99 nt) products are indicated by green and blue arrows, respectively. The
round numbers are listed above the lanes.
e108 Nucleic Acids Research, 2002, Vol. 30 No. 20 PAGE 8 OF 14

9. aptamers in Family 1 are predicted to form a typical stem±loop
structure closed by the requisite C:G base pair. Conversely,
the remaining base pairs in the stem interact non-speci®cally
with positively charged residues in U1A (12,33,39). Because
of this the identity of the base pairs in the stem have been
found to be irrelevant to U1A binding and, in fact, no
particular base pairs predominate in the predicted stem
structures of the Family 1 aptamers. Similarly, the last three
residues (UCC) of the 10 residue hairpin loop of the wild-type
U1 snRNA do not physically interact with the protein (12).
Family 1 aptamers all have three or four dissimilar (non-
conserved) residues in these same positions. The variability in
overall loop length was expected, given that it has previously
been shown that the insertion of a single residue into the loop
has little effect on protein binding (35). Conversely, no loops
shorter than 10 residues were seen. In this regard, it has
previously been shown that a nine residue loop lacking one of
the three otherwise non-conserved residues has 1000-fold less
af®nity for U1A. The removal of another residue decreases
binding an additional 10-fold, while the creation of a seven
residue loop ultimately decreases the Kd by 100 000-fold (35).
In the wild-type 3¢-UTR the canonical AUUGCAC and
loop-closing C:G base pair again form hydrogen bonds and
hydrophobic interactions with U1A (33,34) and, as stated
before, Family 2 aptamers retain all critical sequence and
structural features. In addition, in the 3¢-UTR a single
adenosine residue (A24) on the opposite side of the internal
loop has been shown to form essential hydrogen bonding and
stacking interactions with serine, valine and arginine residues
in U1A (33,34). Aptamers in Family 2 can in fact be folded so
as to present a single adenosine residue opposite the conserved
heptamer.
The wild-type RNA ligands of the U1A protein are known
to bind with high af®nity (Kd values of 10±11±10±8 M,
depending on pH and monovalent and divalent salt concen-
trations) (35). Under our assay conditions, the minimized, 21 nt
wild-type U1 snRNA hairpin II structure forms a complex
with U1A with a Kd of 11 nM. Most aptamers were found to
Figure 5. Progress of automated U1A selection. Binding assays are shown for rounds 0, 6, 12 and 18 of the selection. The ®rst column (light blue) shows the
fraction of RNA captured in a standard binding assay. The second column (dark blue) shows the fraction of RNA captured when a high salt wash was used to
remove non-speci®cally bound RNA.
PAGE 9 OF 14 Nucleic Acids Research, 2002, Vol. 30 No. 20 e108

10. have Kd values in the low nanomolar range (Table 3). Family 1
aptamers, those most akin to hairpin II, have Kd values as low
as 4.5 nM. Interestingly, at least one member of Family 1,
clone 09, has a much higher Kd value (69 nM). This aptamer is
also predicted to form a hairpin-like structure, just as the other
family members are. The fact that its Kd deviates signi®cantly
suggests that subtle changes in the presentation of the
heptamer motif can be extremely important for binding.
Family 2 aptamers bind U1A slightly worse than Family 1,
with the majority aptamer forming a complex with a Kd of
17 nM. As expected, sequences with `near perfect' motifs
bound much less well than sequences that conformed to the
natural ligands.
The selection of the natural binding sequences was not
completely unexpected. A previous manual selection had been
performed against the U1A protein by Tsai et al. (8). The
authors carried out selection experiments starting from three
randomized pools. The ®rst two pools contained the wild-type
Figure 7. (Following page) Sequences of anti-U1A aptamers. Sequences of selected clones from (A) round 6, (B) round 12 and (C) round 18 are shown. The
static (priming) regions are shaded light gray, while the randomized region is in black text. Perfect matches to the U1A snRNA heptamer are highlighted in
gray and bold red; near perfect heptamer sequences are highlighted in gray. In (C), the C:G base pair that closes the U1 snRNA hairpin II loop (Family 1)
and that precedes Box II of the 3¢-UTR U1 mRNA (Family 2) is shown in blue. The adenosine found in the internal loop region of the 3¢-UTR U1 mRNA
(Family 2) is shown in green.
Figure 6. U1A binding elements. (A) U1 snRNA hairpin II; (B) the 3¢-UTR of U1 mRNA. The heptamer sequence critical for recognition by U1A is high-
lighted with a black bar. The 3¢-UTR of U1 mRNA (B) contains a second heptamer sequence (Box I) that contains a base change (AUUGUAC) at the ®fth
nucleotide. The critical C:G base pair that closes stem II and that precedes Box II is in bold. The adenosine found in the internal loop region of the 3¢-UTR is
also in bold. (C) Aptamer Family 1, drawn in a manner similar to (A). (D) Aptamer Family 2, drawn in a manner similar to (B). Residues contributred by the
primer-binding sites are shown as dark yellow.
e108 Nucleic Acids Research, 2002, Vol. 30 No. 20 PAGE 10 OF 14

11. PAGE 11 OF 14 Nucleic Acids Research, 2002, Vol. 30 No. 20 e108

12. U1 snRNA stem structure topped by a randomized loop region
of 10 or 13 nt. The third pool contained 25 randomized nt
¯anked by primer sequences, similar to our N30 pool. Both the
constrained and unconstrained pools generated aptamers
containing the canonical heptamer sequence. How-
ever, the aptamers generated from the third pool by these
manual selection procedures exhibited poor binding to U1A
protein in gel shift experiments (100-fold less af®nity than
with wild-type ligand). In keeping with this observation, the
manually selected aptamers did not mimic the wild-type
sequences as well as those generated by automated selection;
for example, they did not contain the C:G base pair closing the
U1 snRNA loop. Similarly, none of the aptamers selected from
the unconstrained pool was predicted to fold into either U1
snRNA-like or U1A 3¢-UTR mRNA-like structures. While
additional rounds of manual selection might of course have
improved the selected populations, these would have been
prohibitively tedious. The fact that multiple rounds of
automated selection can be carried out within days allows
the sequences, structures and af®nities of aptamers to be ®nely
tuned.
Prospects for automated selection with in vitro generated
protein targets
Overall, the results presented here illustrate a strategy for the
generation of multiple aptamers against a large number of
protein targets and for the acquisition of sequence and
structural data relating to RNA±protein interactions. Based
on the throughputs available with automated selection, the
generation of aptamers against the translation products of all
genes within an average bacterial genome could be completed
within a course of months. Many of the selected aptamers
would of course be useful as reagents alone, for example for
labeling or detecting (1±3) key proteins or even the expression
pro®le of the entire proteome. Nonetheless, the generality of
these methods for multiple different classes of proteins
remains to be seen.
Our results also con®rm that it may be possible to use
in vitro selection to decipher speci®c interactions between
multiple different nucleic acid-binding proteins and their
corresponding cellular RNA sequences. In vitro selection has
previously been used to identify or de®ne a variety of natural
nucleic acid-binding sequences (42). For example, an in vitro
selection experiment that targeted the E.coli methionine
repressor generated DNA aptamers that contained an octamer
sequence found in natural DNA-binding sites (43). Likewise,
in vitro RNA selection experiments have generated aptamers
to T4 DNA polymerase that both recreate a natural binding
element and generate a new element that possesses the same
Kd as the wild-type (44). Similar ®ndings have been observed
during selections against phage MS2 coat protein, phage R17
coat protein and the E.coli special elongation factor SelB
(45±47).
From this vantage, it is interesting to note that our selection
produced two aptamer families that closely corresponded to
the two natural RNA-binding elements of U1A. The fact that
the natural sequences and structures were extracted from a
completely random sequence library is remarkable and has
additional important implications for the speci®city of inter-
actions between U1A and its natural targets. One measure of
speci®city is what set of sequences a given protein will
preferentially recognize. In the case of U1A, the natural
ligands are apparently globally optimal, at least within the
context of a sequence space of roughly 30 residues. Indeed, the
U1A protein can even ®nely distinguish between ligands that
super®cially resemble one another in terms of critical
sequences and structures, since different Family 1 members
can have Kd values that vary by an order of magnitude. These
results imply both that the U1A protein has structured its
RNA-binding domain in such a way that it can reject
sequences and structures that are not exactly like the natural
ligands, and that the natural ligands have been derived by a
search of a sequence space that is much larger than the size of
the genomes in which they are ensconced. This is one of the
®rst examples of natural optimality in a sequence space of this
size.
While other methods for ascertaining natural nucleic acid-
binding sequences are also possible, including genomic
SELEX (48±50), in which libraries are generated directly
from natural sequences, the use of randomized libraries also
allows the identi®cation of unnatural sequences that may have
improved af®nities and greater utility as laboratory reagents.
For example, in vitro selection against the Rev protein of
HIV-1 yielded aptamers that were similar to the wild-type
Rev-binding element (RBE), but bound to Rev 10 times better
(51). In the current instance, despite the fact that the natural
RNA sequences have been exquisitely optimized by natural
selection, automated selection has apparently improved on
wild-type af®nities by a factor of approximately 2.4 (4.5
versus 11 nM).
Table 3. Dissociation constants of
complexes formed with selected
sequences
Numbers under families are clone
numbers from round 18. Binding assays
were performed in triplicate to determine
standard deviations.
e108 Nucleic Acids Research, 2002, Vol. 30 No. 20 PAGE 12 OF 14

13. ACKNOWLEDGEMENTS
We thank Dr David Waugh (NCI±Frederick) for the gift of
pDW363. This work was funded by grants from the Beckman
Foundation, Robert A. Welch Foundation (F-1393), Army
Research Of®ce, (MURI DAAD19-99-1-0207) and NIH-Rev
(AI36083-09).
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