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SELEX
Selective Evolution of Ligands
by Exponential Enrichment
GSH
Dorothee von Laer
In vitro selection, or SELEX, is a technique that allows the simultaneous
screening of highly diverse pools of different RNA or DNA (dsDNA or
ssDNA) molecules for a particular feature.
In 1990, the laboratories of G. F. Joyce (La Jolla), J.W. Szostak
(Boston), and L. Gold (Boulder) independently developed a technique
which allows the simultaneous screening of more than 10exp15
individual nucleic acid molecules for different functionalities. This
method is commonly known as "in vitro selection", "in vitro evolution" or
"SELEX" (systematic evolution of ligands by exponential enrichment).
This novel technique is gaining more and more importance as an
extremely useful tool in molecular biology. With the in vitro selection-
technique large random pools of nucleic acids can be screened for a
particular functionality, such as the binding to small organic molecules,
large proteins or the alteration or de novo generation of ribozyme-
catalysis. Functional molecules ("aptamers" a linguistic chimaera
composed of the latin aptus = to fit and the greek suffix -mer) are
selected from the mainly non-functional pool of RNA or DNA by column
chromatography or other selection techniques that are suitable for the
enrichment of any desired property.
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SELEX schemeDNA library
(1015 random sequences)
24 nt 40 nt 21 nt
Cloning out
Analysis of individualsequences
Increase of stringency
- Addition of tRNA
- Counter-SELEX
The SELEX process is as follows:
In a standard DNA-oligonucleotide synthesizer a starting pool is generated. The
machine synthesizes an oligonucleotide with a completely random base-sequence
which is flanked by defined primer binding sites. In this way, up to 10exp15 different
DNA molecules can be synthesized at once, which is an incredibly complex pool, if
one considers the number of antibodies a mouse can possibly generate between
10exp9 and 10exp11. The immense complexity of the generated pool justifies the
assumption that it contains a few molecules with the correct receptor structure or with
tertiary structures which lead to catalytic activity; these are selected, for example by
affinity chromatographyor filter binding. Because a pool of such high complexity can
be expected to contain only a very small fraction of functional molecules, several
purification steps are usually required. Therefore, the very rare active molecules are
amplified by the polymerase chain reaction (PCR) or in a transcription-based step. In
this way, iterative cycles of selection can be carried out. Successive selection and
amplification cycles result in an exponential increase in the abundance of functional
sequences, until they dominate the population. The method has been applied to a
number of different applications; for example, in vitro selection has proven to be
extremely efficient for the identification of bases which cannot be changed without
loss of function and are important in ribozymes, or in a protein binding site in a (ds or
ss)DNA or RNA molecule. Recently, in vitro selection has been used for the de novo
isolation of catalytic RNAs. These include ribozymes with ligation activity, isomerases
and ribozymes which catalyze the ATP-dependent phosphorylation of RNA
oligonucleotides. The basis for the latter two ribozymes was the isolation of RNAs for
specific binding to small substrate molecules, for which several examples exist. RNA-
and DNA-aptamers have been isolated, which not only bind tightly to proteins, but
also are able to inhibit their biological activity.
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SELEX Process RNA/DNA
Compared to DNA aptamers, the selection of RNA aptamers by SELEX
requires two additional reactions: reverse transcription of the selected
RNAs and an in vitro transcription of the cDNA.
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T7 promotor5 bp N30 random29 bp 20 bp
For primer Rev primer
DNA library
(101 bp)
In vitro
transcription
N30 random29 nt 20 ntRNA library
(79 nt)
RT
( + Rev primer)
N30 random29 nt 20 nt
PCR
( + For and Rev primer)
T7 promotor5 bp N30 random29 bp 20 bp
The templates and primers used for transcription, reverse transcription
and PCR reactions in the SELEX process are shown for RNA
aptamers.
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Multiple
accessories
Integrated
plate reading
Filtration unit for
DNA purification
Rapid screening
High capacity using
the stacker carousel
Introducing
Biomek 2000(Beckman Coulter)
A SELEX robot can reduce the work load for SELEX effectively.
Classically, 2-3 days are required per SELEX round and 10 rounds
must be performed at least to obtain effective binders. Using the robot,
10 rounds of selection against 8 targets in parallel can be performed
within 4-5 days.
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Special forms of SELEX
Selection of Spiegelmers
(select D-RNA/D-DNA (natural) against
enantiomer e.g. D-Peptide-> L-RNA/L-DNA is
Spiegelmer against natural L-peptid)
Selection of short aptamers by tailored SELEX
Spiegelmers are mirror-image, high-affinity oligonucleotide ligands
composed of L-ribose or L-2'-deoxyribose units. The chiral inversion
results in high stability in plasma compared with natural D-
oligonucleotide ligands, aptamers, suggesting that Spiegelmers may
display favorable in vivo behavior and present future potential for
therapeutic and diagnostic applications. Spiegelmers thus offer a
promising alternative to aptamers, the limited in vivo stability of which
continues to be a major obstacle to clinical development despite
extensive efforts to improve the structure of the oligonucleotide
backbone.Spiegelmers can fold into distinct three-dimensional
structures generating high-affinity ligands that can be selected against
defined pharmacological targets. High-affinity Spiegelmers with the
desired target-binding properties can be identified by using an
adaptation of the SELEX (systematic evolution of ligands by exponential
enrichment) procedure. Because L nucleic acids are not compatible
with SELEX because of the enantio specificity of the enzymes used for
amplification, a "mirror-image" SELEX approach is used. The first step
is to select an aptamer against the enantiomeric form of the natural
target. After trimming to the minimal binding motif, the equivalent L form
of the aptamer, the Spiegelmer, then is synthesized, and because of the
reciprocal chirality, this Spiegelmer binds with high affinity to the natural
target.
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Vater, A. et al. Nucl. Acids Res. 2003 31:e130; doi:10.1093/nar/gng130
Tailored SELEX ommiting primer binding sites in selection
Tailored SELEX allows the direct and rapid isolation of target binding
RNA sequences that only require 10 fixed nucleotides in addition to
the random region. This novel procedure relies on customized primers/a
dapters that are added by ligation before and removed within the
amplification processes by alkaline fission.
(A) Cartoon of the RNA library and the double-stranded adapters each containing a ligate and an oligonucleotide bridge, before the ligation of the primer binding sites. The library consists of a randomized region that is flanked by 4 and 6 nt long stretch
es of fixed sequence (green). They serve as hybridization sites for the bridging oligonucleotides of the pre-annealed double-stranded adapters. The forward ligate contains a T7 RNA polymerase promoter at its 3' end. Reverse bridge 1 is also used as a PCR
reverse primer. Two nucleotides in the reverse bridge 1 are uridines (U) which allow for primer removal under alkaline conditions. Forward bridge and reverse ligates contain a 3' terminal 2'-3'-dideoxynucleotide (3'H) to prevent them from mispriming in th
e PCR. (B) Up to 50% of all run-off transcripts contain a non-templated nucleotide (N) at their 3' ends (red). In order to ligate these species, an alternative adapter 2 was designed. It consists of a reverse ligate 2 that lacks the 5' terminal adenosine
(A) and a reverse bridge 2 that offers the universal base inosine for hybridization opposite to the additional nucleotide. Thus, the overall length of the library does not increase.
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Separation of Bound from Unbound RNA
Column
Beads
Filter binding
Target expressed on whole cell surface
Gradient centrifugation
Capillary electrophoresis
Affinity Maturation
Biacore
Competition with known ligand, antibody
Gel shift
9
Copyright restrictions may apply.
Vater, A. et al. Nucl. Acids Res. 2003
Example of a SELEX Experiment
The histogram shows the course of the in vitro selection. The
fraction of the RNA pool eluted from the underivatized streptavidin
or neutravidin matrix (yellow bars) and from the identical matrix after
capturing RNA:peptide (calcitonin gene-related peptide 1 (-CGRP))
complexes from a solution (green bars) with the indicated peptide
concentration (red triangles) is shown. Starting from round 6,
selection was usually performed at three different peptide
concentrations. Only the data of the minimal successful peptide
concentration are shown.
10
Copyright restrictions may apply.
Vater, A. et al. Nucl. Acids Res. 2003
Aligned sequences from the CGRP binding RNA pool after selection
round 15. The numbers indicate the individual sequence’s
frequency of occurrence. The fixed sequence parts were shaded in
gray. Four different conserved motifs were identified as indicated by
the background colors. The blue motif was found to occur as a
24mer or in part as an 11mer if it is flanked by the magenta-colored
16 nt long split-motif. The blue motif appeared close to the 5' end, in
the middle or close to the 3' end of the randomized region. While the
green and magenta motifs were unique to the in vitro selection at
37°C, the blue and yellow motifs were also frequent in room
temperature selections that were carried out in parallel (sequences
not shown). The sequences within each group seem to have partly
arisen from an identical ancestor sequence with Taq polymerase-
induced mutations.
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Chemical modifications of RNA aptamers and their purpose.
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2‘-F- versus 2‘-NH2-pyrimidines
NH2 more flexible but
F structures more stable,
have higher affinities and
are more efficiently synthesized...thus more economical
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Interaction with Target
•Shape complementarity
•Hydrogen bonding
•Electrostatic interactions
•Stacking interactions
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Binding of apatamers to aromatic ligands:
Theophyllin FMN AMP
TheophyllinFMN
AMP-DNA
Aptamer
AMP-RNA
Aptamer
Aptamers that are highly specific for theophyllin were selected that do
not crossreact with caffein. Theophyllin is a drug used in Asthma
therapy. The aptamer is used to determine plasma concentrations in
treated patients.
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RNA-Aptamer with
an R-rich peptide
from HIV rev protein
Alpha-helix in widened
deep groove of RNA
RNA aptamer bound to
bacteriophage M52
coat protein
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Structure of aptamers
Enzymatic probing
Crystallographic structure
NMR
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ANTU K. DEY et al. RNA 2005; 11: 873-884
FIGURE 1. Enzymatic probing, RNA
footprinting and solution structure of
aptamer B40 and B40t77
Nuclease:
T1 5‘ G ssRNA
S1 ssRNA
V1 ds RNA
Chemical modification
DMS
A, G and C not in pairs
CMCT
G and U not in tertiary interactions
RT stops at modified nucleotide
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Application
•Diagnostics
•Therapy
•Biotechnology
•As catalysts: Ribozymes regulated by
cofactors
•To regulate translation by inserting
into mRNA (riboswitch)
In molecular biology, a riboswitch is a part of an mRNA molecule that can directly bind
a small target molecule, and whose binding of the target affects the gene's activity.
Thus, an mRNA that contains a riboswitch is directly involved in regulating its own
activity, depending on the presence or absence of its target molecule. Riboswitches
are conceptually divided into two parts: an aptamer and an expression platform. The
aptamer directly binds the small molecule, and undergoes structural changes in
response. These structural changes also affect the expression platform, which is the
mechanism by which gene expression is regulated.
Expression platforms typically turn off gene expression in response to the small
molecule, but some turn it on. Expression platforms include:
* The formation of transcription termination hairpins
* sequestering the ribosome-binding site, thereby blocking translation, and
* self-cleavage (i.e. the riboswitch contains a ribozyme that cleaves itself in the
presence of sufficient concentrations of its metabolite).
Most known riboswitches occur in eubacteria, but functional riboswitches of one type
(the TPP riboswitch) have been discovered in eukaryotes. Sequences similar to
known TPP riboswitches have also been found in archaea, but are not experimentally
verified.
Although the genetic pathways in which riboswitches are involved have been studied
for decades, the existence of riboswitches has only recently been found. This
oversight may relate to an assumption that genes are regulated by proteins, not by the
mRNA transcript itself. Now that riboswitches are a known mechanism of genetic
control, it is reasonable to speculate that more riboswitches will be found.
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Diagnostics
•ELISA
•FACS
•Sensors
•Fluorescence polarization
•Capillary electrophoresis
•Eastern blot
•Molecular beacon
Aptmers can be broadly used for diagnostic purposes as reviewed in
Tombelli et al., Biosensors and Bioelectronics 20: 2424, 2005.
The concept of molecular movement and rotation is the basis of
fluorescence polarization. By using a fluorescent dye to label a small
molecule, its binding to another molecule of equal or greater size can
be monitored through its speed of rotation.
Ribozymes whose activity is switched on or off by the presence of a
specific target. These unique ribozymes, known as RiboReporter™
Sensors act as reporter molecules in that they directly couple molecular
detection to the triggering of a chemical reaction. The combination of
these properties in a single molecule makes them powerful tools for a
wide range of applications.
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Molecular beacons, a nucleic acid motif that possesses a stem-loop structure, have been
exploited to ®nd a complementary target sequence (Tyagi & Kramer 1996). Molecular beacons
essentially contain two structural components, a loop and a stem. The loop sequence serves as
a probe, which is complementary to the target sequence, and the annealing of two
complementary arm sequences that are flanked by the probe sequence forms the stem. One
fluorescence and one non-fluorescent quencher are linked covalently at each end of the arm.
The stem of the beacons brings the fluorophore and quencher into close proximity to each
other, resulting in zero fluorescence. When the molecular beacon encounters a target molecule,
it forms a probe±target hybrid that is stronger and more stable than the stem in the hairpin. The
resulting conformational change in the stem-loop oligomer forces the sequence of the two arms
apart, thus permitting the fluorophore to fluoresce. Consequently, molecular beacons allow a
real-time detection of speci®c nucleic acids without interrupting their reactions which is also
applicable to living cells (Piatek et al. 1998; Matsuno 1998; Sokol et al. 1998). However, the
most signi®cant parameters affecting the conformational switch in the molecular beacon (Tyagi
& Kramer 1996), appear to be the length of the arm and probe sequences. In addition, the
probe sequence (target sequence) should be at least twice the length of each arm sequence in
order to render the conformational change (Tyagi & Kramer 1996). Thus, the speci®city of the
molecular beacons depends entirely on the target sequence (loop of the molecular beacon).
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Aptamers versus Antibodies
LimitedLongShelf-life
IrreversibleReversibleDenaturation
BiologicalSyntheticProduction
Only physiologicalCan be selectedConditions of
interaction
yesnoSensitivity to heat
Immunogenic
/non-toxic
Potentially anyTarget
In vivoIn vitroIsolation
AntibodyAptamerCharacteristic
Aptamers have several advantages over antibodies. They can
potentially be generated against any target, under any condition, they
can be produced synthetically and are highly stable.
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Antibody-aptamer size
comparison
23
Therapeutics
• Decoys
• Block molecular interactions
• Block enzyme function
• To target therapeutic molecules
24
Decoy
Example for a decoy: The TAR decoy competes with the HIV TAR RNA
for binding to the HIV transactivator Tat protein and thereby reduces
Tat dependent viral transcription. Tar decoys thus inhibit HIV
replication.
25
gp120gp41
Zellmembran
CD4Co-R Zelle
Außen
gp41
Virus
gp120
Bindung von HIV an die Zielzelle
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CXCR4
or CCR5
gp120gp41
gp41
Virus
gp120
Konformationsänderung von HIV gp120
-> Bindung an Co-Rezeptor CCR5/CXCR4
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Cell
HIV gp41 Fusionspeptid dringt in die
Zellmembran ein
gp41Virus
gp120
Membrananker
C-heptad repeat
N-heptad repeat
Fusionspeptide
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Zelle
Bildung des gp41 6-Helix-Bündels aus den
N- und C-heptad repeats Sequenzen
gp41
Virus
gp120
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Cell
Fusion der viralen und zellulären Membranen
gp41
Virus
gp120
30
gp120gp41
Zellmembran
CD4Co-R Cell
gp41Virus
gp120
RNA-Aptamer-Targets
Inhibition der Bindung an CD4
Aptamere gegen die
CD4 Rezeptor-
Bindungsdomäne
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gp120gp41
CD4Co-R
gp41
Virus
gp120
Inhibition der Co-Rezeptorbindung
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Cell
Gp41:
Virus
gp120
C-heptad repeat
N-heptad repeat
Inhibition der Bildung eines 6-Helix-Bündels und damit der Fusion
33
The first aptamer approved by the FDA as a drug: Macugen for
treatment of macula degeneration (AMD).
34
Copyright restrictions may apply.
The Eye Diseases Prevalence Research Group, Arch Ophthalmol 2004;122:477-485.
Causes of blindness (best-corrected visual acuity <6/60 [<20/200] in the better-seeing eye) byrace/ethnicity
AMD is the most frequent cause of blindness is developed countries.
35 36
Age-related macula degeneration (AMD)
Dry90 % of AMD but
10 % of AMD-associated blindness
Wett form10 % of AMD but
90 % of AMD-associated blindness
There are 2 types of macula degeneration: a dry and a wett form. The
wett form is associated with neovascularisation (VEGF dependent) and
more frequently with blindness.
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Neovascularisation in AMD
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VEGF
•Central positive regulator of angiogenesis
•Endothelial cell mitogen+chemoattractant
•Induces vascular permeability and angionenesis
•Receptors: Flt-1, Flk-1
•Homodimer, disulfide-linked,
•4 isomers by alternative splicing (121, 165,189,206)
•Promotes tumor vasculature and thus tumor growth
•Promotes vision loss by neovascularisation in AMD
and diabetic retinopathy
•Possible role in rheuma and psoriasis
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