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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

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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.

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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

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Therapeutics

• Decoys

• Block molecular interactions

• Block enzyme function

• To target therapeutic molecules

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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.

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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

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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

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The first aptamer approved by the FDA as a drug: Macugen for

treatment of macula degeneration (AMD).

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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.

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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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