This presentation include- "Introduction, Method of preparation, advantages, Disadvantages and application of Aptamer"

1. Seminar
on
APTAMERS
Presented by
Geetanjali
(M.Pharm 1st Year)
Enrollment No:18022020004
Department of Pharmacy
School of Medical and Allied Sciences
Galgotias University
Yamuna Expressway, G.B. Nagar (U.P.)

2. CONTENTS
• Introduction
• Properties
• Aptamers production
• Classification of Aptamers
• Aptamers selection methods
• Comparison
• Advantages
• Disadvantages
• Applications
• Conclusion
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3. INTRODUCTION
In, Latin aptus/apto – to fit,
and
In, Greek meros – part (Smallest Unit Of Repeating Structure)
• Aptamers are oligonucleotide or peptide molecules that bind to
specific target molecule.
• Aptamers are usually created by selecting them from a large
random sequence pool, but natural aptamers also exist in
riboswitches.
• Aptamers can be combined with ribozymes to self-cleave in the
presence of their target molecule.
• They range in size from 20 to 80 bases (6-26kda).
• They are synthetic single-stranded DNAs or RNAs, which bind to
target molecules with high affinity in three-dimensional shapes.
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4. APTAMERS PRODUCTION
• Aptamer are produced by an in vitro selection or systematic
evolution of ligand by exponential enrichment(SELEX).
• It will produce ssRNA with specific binding target.
• This is an iterative process of binding, partitioning,
amplifying novel nucleic acids and regeneration.
• The first step is to synthesize a large pool of nucleic acid
molecules made from average of 15-40 bases of random
sequences that are flanked by primers 5’ and 3’ end.
• The pool of DNA is transcribed into an RNA pool and it
subsequently exposed to the target ligand of interest.
• Affinity column chromatography removes unbound sequences
identify the strongest binding sequences and bound sequences
are eluted and amplified by RT-PCR
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5. 5

6. CLASSIFICATION OFAPTAMERS
BASED ON DIFFERENTTECHNIQUES
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7. Aptamer Selection Methods
Capillary
Electrophoresis-
Based SELEX
Conventional
SELEX
Magnetic Bead-
Based SELEX
Cell -SELEX
In Vivo SELEX
Nitrocellulose
Membrane
Filtration-Based
SELEX
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8. CONVENTIONAL SELEX
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9. A chemically synthesized oligonucleotide library is usually
used for aptamer selection. This library contains up to 60
oligonucleotides in random region, which are flanked by short
constant regions, usually used to anneal primers during PCR.
Several selection steps are introduced during conventional
SELEX depending on the type of the desired aptamer.
In the case of DNA aptamers, a chemically synthesized DNA
library is incubated with target molecules. Unbound molecules
are removed and the target/DNA complex is split. Released
DNA sequences are amplified by PCR and additional round of
selection is performed. Potential aptamers, obtained from a
number of selection rounds, are sequenced and their binding
kinetics are evaluated by various methods.
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10. In the RNA aptamer selection, additional steps of in vitro
transcription and reverse transcription are introduced. Thus, a
chemically synthesized DNA library is transcribed into a RNA
library, which undergoes selection similar to DNA aptamers.
Distinctly, RNA sequences released from their target are
reversely transcribed into DNA and amplified by PCR.
Multiplied DNA molecules are transcribed back into RNA and a
new selection round starts over. Up to 20 rounds of selection are
usually performed to enrich aptamers with a high target affinity.
Conventional SELEX is a well-established and effective method
but due to its large time and labor consumption, continuous
development of alternative methods for aptamer selection has
been inevitable.
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11. CAPILLARY
ELECTROPHORESIS-SELEX
 This method is separation of an
aptamer–target complex from
unbound oligonucleotides
according to their electrophoretic
mobility.
 The oligonucleotides bound to the
target have a lower mobility than
free oligonucleotides, which pass
through a capillary into a waste.
 Then, pressure is applied to collect
the aptamer/protein complex. Many
aptamers selected by CE-SELEX
have been published so far. 11

12. They include the following: aptamers against IgE , neuropeptide Y,
human immunodeficiency virus reverse transcriptase (HIV-1 RT, ssDNA
aptamer), ricin toxin (ssRNA), protein kinase C δ and anthrax protective
antigen (ssDNA modified into nano aptasensor).
Recently, a modification of CE-SELEX led to invention of micro free-
flow electrophoresis (μFFE) .This method brought a dramatic increase
in aptamer partitioning efficiency in comparison to CE- and
conventional SELEX, already after the first round of aptamer
selection.
Other techniques based on capillary electrophoresis are kinetic capillary
electrophoresis (KCE) methods of aptamer selection. These include
NECEEM (non-equilibrium capillary electrophoresis of equilibrium
mixtures) ,ECEEM (equilibrium capillary electrophoresis of equilibrium
mixtures) and SweepCE (sweeping capillary electrophoresis). 12

13. MAGNETIC BEAD-BASED
SELEX
13

14. A SELEX method employing magnetic beads for
immobilization of the target was published in 1997 . In this
method, called magnetic bead-based SELEX, the target
protein was immobilized on magnetic beads, incubated with an
oligonucleotide library and resulted in a target–aptamer
complex that was separated from unbound oligonucleotides by
a magnetic separator.
After the first round of selection, ssDNA was amplified by
PCR and labeled using fluorescein-modified primers for
quantification of selected aptamers in further selection rounds.
The obtained oligonucleotides are then cloned and sequenced.
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15. CELL- SELEX
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16. All previously described SELEX methods are based on knowledge of
the target for aptamer selection.
The only exception is cell-SELEX. This well-developed method does
not require prior target knowledge. Whole cells, both eukaryotic and
prokaryotic, can be used to generate highly specific aptamers using this
technique.
As in conventional SELEX, an oligonucleotide library is incubated with
the targets, which are whole cells in the case of cell-SELEX. The
unbound oligonucleotides are washed out and aptamers are separated
from the target and amplified by PCR.
Cell-SELEX may require up to 35 selection rounds but most commonly
8–10 rounds are applied in order to obtain aptamers with high affinity.
A series of aptamers against various cancer cell types were generated by
this method.
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17. Advantages ofAptamers
• Easier and more economical to produce.
• Compared to antibodies, toxicity and low immunogenicity of
particular antigens do not interfere with the aptamer selection.
• Aptamers are capable of greater specificity and affinity than
antibodies.
• Aptamers can easily be modified chemically to yield
improved, custom tailored properties.
• Aptamers can specifically bound to either small molecules and
complex multimeric structures.
• Improved transport properties allowing cell specific targeting
and improved tissue penetration.
• Aptamers are much more stable at ambient temperature than
antibodies.
• Ability to inactivate proteins, without altering genetic
material.
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18. Disadvantages ofAptamers
• Lower levels of
affinities than
antibodies.
• Aptamers will
not bind to some
target molecules.
• Aptamers
identification is
expensive and
labour intensive.
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19. APPLICATIONS
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20. COMPARISON
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21. Progress of aptamers for diseases’
therapy in on-going or completed clinical
trials.
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22. • Various aptamer-based drug delivery systems such as aptamer-
chemotherapy agents, aptamer-siRNA/shRNA/miRNA,
aptamer-antibody, aptamer-enzyme and aptamer-nanoparticles
have been established to specifically deliver the drug to the
expected sites, therefore reducing the possibility of side effects
caused by the off-target effects.
• Nonetheless, the attractive advantages of aptamers over
antibodies still bring broad prospects for aptamers development.
With many advances in place, several aptamers are now in
clinical trials.
• In summary, with the advances of SELEX technology, new
aptamers are being developed in a more efficient way with less
cost. It can be expected that more aptamers can be applied to
diagnostic and therapeutic purposes in the near future.
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23. References
• Muir, P.; Li, S.; Lou, S.; Wang, D.; Spakowicz, D.J.; Salichos, L.;
Zhang, J.; Weinstock, G.M.; Isaacs, F.; Rozowsky, J.; et al. The real
cost of sequencing: Scaling computation to keep pace with data
generation. Genome Biol. 2016, 17, 53. [CrossRef] [PubMed]
• Sundaram, P.; Kurniawan, H.; Byrne, M.E.; Wower, J. Therapeutic rna
aptamers in clinical trials. Eur. J. Pharm. Sci. 2013, 48, 259–271.
[CrossRef] [PubMed]
• Zhou,J.;Rossi,J.Aptamers as targeted therapeutics: Current potential
and challenges. Nat. Rev. DrugDiscov. 2017, 16, 181–202. [CrossRef]
[PubMed]
• Yu, Y.; Liang, C.; Lv, Q.; Li, D.; Xu, X.; Liu, B.; Lu, A.; Zhang, G.
Molecular selection, modification and development of therapeutic
oligonucleotide aptamers. Int. J. Mol. Sci. 2016, 17, 358. [CrossRef]
[PubMed]
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24. Thank you
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