In the name of God
Aptamer in-vitro selection
SELEX & Non-SELEX
Maryam Tabarzad
Shahid Beheshti University of Medical Science
1392
History
Aptamers are synthetic, highly structured, single stranded DNA/RNA ligands
Term “Aptamer”(Ellington&Szostak 1990) : aptus (to fit) + mer (oligo)
The first SELEX experiment on single- stranded oligonucleotides was
published by Tuerk and Gold in 1990
'Systematic evolution of ligands by exponential enrichment' (SELEX)
A protocol in which vast libraries of single-stranded oligonucleotides are
screened for desired activities
Systematic Evolution of Ligands by Exponential enrichment
SELEX components
• Oligonucleotide random pool
• DNA/RNA
• Modified nucleic acids
DNA or RNA
• RNA : in vitro transcription
• ssDNA:
Asymmetric PCR
Biotin-streptavidin separation
Lambda exonuclease digestion
Size separation on denaturing-urea PAGE
SELEX components (cont.)
• Target
• Small molecules
• Bimolecules
• Whole cells
• Whole organisms
SELEX components (cont.)
• Separation techniques
• Filtration (nitrocellulose, Dialysis)
• Chromatography (size exclusion, Affinity)
• Electrophoresis (PAGE, Agarose, CE)
• Magnetic beads
• Precipitation (Immunoprecipitation, centrifuge)
• SPR
• AFM
Targets of selection
Negative SELEX
To exclude adsorbed ssDNA or RNA by the matrixes used for immobilizing
targets with the purpose of gaining aptamers that can only adsorb the targets
Counter SELEX
• Alike to the negative SELEX
• In order to get aptamers with stronger specificity
• Excluding ssDNA or RNA molecules with coaffinity to the similar
molecules of the targets.
• Established by Jenison and his fellows(theophylline , caffeine )
Subtractive SELEX
• The same purpose as counter SELEX
• To improve the selectivity of aptamers
• The main difference between them lies in the targets of the process.
• The targets of complex targets
• Eliminates the ssDNA or RNA sequences that can bind the no purpose part of
the complex target.
• by Chinese researcher Wang (capability to distinguish differentiated PC12
cells from normal PC12 cells)
Toggle SELEX
• A method to deal with several kinds of targets at the same time
• The different rounds of the screening cycles focus on different
targets according to the strategy during this toggle SELEX.
TECS- SELEX
• Target Expressed on Cell Surface-SELEX
Blended SELEX
• Other molecules, which can lead the oligonucleoride chain to the
specific region of the target, are mixed to the screening pool.
• This method was established by Smith first, For drug discovery
• They connected valyl phosphonate moiety (valP, an inhibitor of
human neutrophil elastase) with the 5’ linker of a DNA splint
oligonucleotide, which can combine with the end of the RNA pool
according to the principle of complementary base pairing.
• This inhibitor could be joined to the random sequence to form a
“blended pool”
Conditional SELEX
• Method for producing nucleic acid ligands that generate a signal,
or cause a decrease in the level of a signal, in the presence of a
target molecule or an environmental stimulus.
• To measure the concentration of a target molecule or detect and
quantitate an environmental stimulus.
Regulated aptamers
Mirror-image SELEX
• The improvement of their stability and half-lives of DNA
and RNA in vivo becomes the key point
• Stable aptamers can be obtained by Spiegelmer
Technology.
• Screens aptamers of chiral target from dextrorotatory
oligonucleotide pool
• Then, the corresponding levorotatory oligonucleotide,
called Spiegelmers, is synthesized
Spiegelemer
• Spiegelmers have a good stability in vivo for not being able to
be degraded by ribonuclease.
• This method only suits for peptides and small molecules with
optical activity. When targets do not have optical activity, this
method could not be used anymore.
• Spiegelmer cannot be amplified by PCR, it loses the
advantage to be quickly obtained plentiful of aptamer in vitro.
Random oligonucleotide Pool
Photo SELEX
• A screening method that uses the photosensitive nuclear acids’ ability of
covalent cross-link with other molecules under a certain wavelength’s light
The selectivity of the aptamers would be higher
The false positive of this method is also higher
• 5-IU or 5-BrdU is mixed into the screening pool, then the pool incubates
with the target, the mixture is irradiated by ultraviolet radiation to make the
cross-link reaction happen
•
The wavelength of the UV radiation and the irradiation time should be
optimized to ensure that only specific sequences could react with the targets.
• This method was first used in 1995 by Jensen, who obtained aptamers for Rev
of HIV-1 by using 5-IU
Genomic SELEX
• Uses the whole genome of a certain organism as the screening
pool
• Bioactive molecules as the targets
• Great potential in the research of the interaction of bioactive
molecules and nuclear acids
• Study the regulation networks between protein and nuclear acid in
proteomics
• Include all the gene sequences and the intron sequences of the
genome
cDNA-SELEX
• At 1995, another library modification strategy was
proposed by Dobbelstein et al.
• use of total cell RNA from Human B-cell lymphoma
• Revealed three sites on 28S ribosomal RNA that have the
potential to interact with L22
In Vivo SELEX
• In 1997 the first report of a selection made inside
mammalian cells was done by Coulter et al.
• uses transient transfection and an iterative procedure to
enrich RNA-processing signals in culture
• To find splicing enhancers sites
Expression Cassettes SELEX
• Peviously isolated aptamer to the E2F1 transcription
factor was coupled to a Pol III promoter as an expression
unit (or cassette) in a plasmidic DNA.
• The construct contained a promoter, a tRNA sequence and
the aptamer flanked by randomized regions.
Expression Cassettes SELEX
• When the transcript was generated, tRNA structure stabilized the
aptamer and the randomized stretches formed a stem flexible
enough to allow the formation of the proper configuration for
target recognition.
• This expression cassette yielded RNAs that bind E2F with high
affinity without sacrificing its structure and which can be stably
expressed at high levels in mammalian cells
Multiple pools SELEX
• A method that combines aptamers from different pools via
a certain way
• Screens the combinational production again in order to
obtain multiple functional aptamers
•
Presently, two modes have been established
Chimeric SELEX
• Burke fused pairs of aptamers previously selected and the results
show that the binding ability of the new aptamers to both the
targets were reduced.
•
Applying dual selection pressure to recombined populations
yielded the combinations that were best suitable for binding both
targets.
• The method can generate dual-function aptamers for a wide
variety of applications, including catalysis, novel therapeutics, and
studies of long-range RNA structure
Multi Stage SELEX
• To study of binding mechanism of aptamers and their targets
• Two nucleic acid pools of N40 and N60 were founded to screen aptamers for
cibacron blue and cholic acid
•
•
•
•
•
Stage one starts on the selection of parental aptamers from randomized libraries.
Stage two, a counter-selection step is used to avoid cross-reaction between each
selection target (cibacron blue and cholic acid).
Stage three, obtained aptamers are fused to each other and reselected to isolate
the most affinity allosteric pairs.
Stage four was to separate binding regions and returned to the counter-selection
like stage two.
In stage five, new allosteric-DNA combinations are re-joined and re-selected to
later be cloned and characterized
Tailored SELEX
• Classical screening of aptamers must truncate the primers after sequencing
and this must affect the selectivity of the aptamers, especially the aptamers
with short random areas.
• This technique decreases the number of the oligonucleotides of the primers
according to the principle of complementary base pairing.
• At the time of amplification, primers can be added to both the ends by a
bridge sequences; after the amplification, these primers can be eliminated by
alkali and the new pool for the next cycle is still with short stable sequences.
Primer-Free SELEX
• Wen used a genomic library derived from the bacteriophage, and
the gene 5 protein (g5p) from the phage as the target protein to obtain
aptamers by means of primer free SELEX.
• Primer sequences are removed from the genomic pool before incubation of
the target protein and are then regenerated to allow amplification.
•
A key step in the regeneration of primer-annealing sequences is to use
thermal cycles of hybridization-extension, with the sequences from unselected
pools as templates.
• Pan et al reported their work about minimal primer and primer-free SELEX
protocols for screening of aptamers from random DNA libraries as well.
Signaling Aptamers
• Basic design of a SELEX for aptamer beacons starts with a library
that had been amplified using a 5′-labeled primer (fluorescein)
• Then hybridized with a capture oligonucleotide that is biotynilated
on the 3′-end and coupled to a particular quencher in the 5′-end.
• Hybrids are recovered using streptavidin beads and mixed with the
target
• In such way that only those sequences forming specific
interactions are released and dequenched.
• Each aptamer has the property of emitting a signal proportional to
target concentration.
Partitioning Methods
FluMAG SELEX
• Stoltenburg et al. 2005
• Used fluorescent labeling and an target immobilized over
magnetic beads
• The possibility of applying them as biosensors useful in
clinical approaches
Deconvolution SELEX
• Gold et al. 1998
• Red Blood Cells
• Specific aptamers to cell surface markers
MonoLex
• affinity chromatography followed by
physical fragmentation of the resin
column
• Nitsche et al.
• Complete Vaccinia virus particles
CE SELEX
• Capillary electrophoresis has been high efficiency and widely used in the
analysis of nuclear acids and proteins.
• Capillary electrophoresis was used in the screening of aptamers by Mendonsa
and Bowser firstly at 2004
• The reasons for the high efficiency of CE SELEX are as follows:
first, random sequences with stronger capacity will be obtained in each cycle because of
the high efficiency of capillary electrophoresis
second, the target in this method is in solution, without the interruption of the matrix, the
requirement of negative SELEX will be eliminated
NanoSelection® (nM-AFM SELEX)
• Atomic force and fluorescence microscopy were
combined with small copy number PCR by Peng et al. in
2007
• The possibility to isolate individual aptamers in a single
selection cycle
Microfluidic chips
• A little sample of target-DNA
mixture (up to 5 nL) was injected
into a capillary and separated
with a high voltage setting the
bases of microfluidics SELEX
• Micro-magnetic device (MMS)
SPR
Surface Plasmon
Resonance
Flow cytometery
Cell SELEX
Non-SELEX
• High efficient and fast method for aptamers’ screening.
• The key point of this method is that it omits the
magnification step by PCR.
• The random sequences with binding ability separated
from the previous circle are used directly in the next one.
• The screening process claims higher efficiency of the
separation step, while capillary electrophoresis is a good
choice.
NECEEM
•Nonequilibrium capillary electrophoresis of equilibrium
mixtures is a new separation-based affinity method.
Automated SELEX
• Cox set up a robotic work station configuration based on an augmented
Beckmanu Biomek 2000 Pipetting robot for screening aptamers in 1998 and
obtained aptamers for lysozyme
•
The described robotic work station can carry out eight screenings in parallel
and will complete approximately 12 rounds of selection in 2 days.
• Furthermore, the automatic work station was improved by containing a part of
the generation of protein targets directly transcribed and translated from the
respective gene in vitro on the robotic work station.
•
•
This should further accelerate aptamer screening for proteins and increase the
utility of aptamers as reagents in proteome analysis
Half automated SELEX
• As the screening conditions are not the same for all the aptamers, the
automated system should be designed with some flexibilities such as the
adjustment of the buffer components, the change of the time of incubation,
and the optimization of the conditions for PCR.
• The half-automated screening platform developed by Eulberg in 2005 can
meet the claims above to some degree.
• This system based on Amp 4200E (MWG Biotech Ebersberg, Germany) coworked with ultrafiltration, fluorescence detection, and semiquantitative PCR.
With this system, they obtained RNA aptamer for substance P
Bioinformatics Approaches for SELEX
• In silico analysis has also been used to study the behavior
of nucleic acid populations during SELEX