The document discusses aptamers, which are oligonucleotides or peptides that bind to targets with high affinity and specificity due to their unique three-dimensional structures. It describes how aptamers are produced through an in vitro selection process called SELEX and classified based on their structure and selection technique. The document also compares aptamers to antibodies and outlines several applications of aptamers in therapeutics, drug delivery, bioimaging, diagnostics, and more.

2. Introduction
Properties
Aptamers production
Classification of Aptamers
Aptamers selection methods
COMPARISON
Advantages
Disadvantages
Applications

3. Aptamers are oligonucleotides, such as ribonucleic acid (RNA) and
single-strand deoxyribonucleic acid (ssDNA) or peptide molecules that
can bind to their targets with high affinity and specificity due to their
specific three-dimensional structures.
Especially, RNA and ssDNA aptamers can differ from each other in
sequence and folding pattern, although they bind to the same target.
It is derived from
Latin word, aptus/apto – to fit and
In Greek, meros – part (Smallest Unit Of Repeating Structure)
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).

4. • 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 pool of DNA is transcribed into an RNA pool and it subsequently
exposed to the target ligand of interest.
• And by Affinity column chromatography unbound sequences are
removed. the strongest binding sequences are identified and bound
sequences are eluted and amplified by RT-PCR

5. SELEX STRATEGY

6. CLASSIFICATION OF APTAMERS BASED ON DIFFERENT TECHNIQUES
Aptamers
Aptamer
SELEX
Aptamer-Small
molecule
conjugated system
Cytotoxic
drug
Linkers
Chemical
Labile
linkers
Acid
cleavable
Disulfide
linkers
Enzyme
labile linkers
Peptide
linkers
Beta
Glucuronide
linkers
Non-Labile
Linkers
Aptamer-NPs
conjugated
system
Inorganic
nanomaterial
Gold NPs
Nanoscale
Iron oxide
Mesoporou
s silica NPs
Quantum
Dots
Organic
Nanomaterials
Liposomes
PLGA(Poly Lactic
Co-Glycolic Acid)
Polymer micelles
Dendrimers
Serum Albumin NPs
DNA NPs

7. CLASSIFICATION OF APTAMERS BASED ON STRUCTURE
APTAMERS
NUCLEIC
ACID
RNA DNA XNA
AMINO
ACID
PEPTIDES AFFIMERS

8. Aptamers selection methods
Capillary
Electrophoresis based
SELEX
Conventional In
Vitro SELEX
Affinity chromatography and
Magnetic Bead based SELEX
Cell SELEX
Nitrocellulose
Membrane Filtration
based SELEX
Microfluidic
based
SELEX

9. A. CONVENTIONAL in vitro SELEX
SELEX or in vitro selection is a technique used to isolate aptamers with high affinity for a
given target from approximately 1012–1015 combinatorial oligonucleotide libraries.
In general, the SELEX process is comprised of three steps that are repeated in order to
search for nucleotides that are better able to bind to the target.
1. In the first step (library generation), a library, except the initial compound in the library,
is converted into single-strand nucleotides that consist of random sequence regions,
usually 30–40 mers, flanked by the primer binding site.
2. In the second step (binding and separation), the target-bound library components are
separated from the unbound components.
3. Finally, in the third step (amplification), the target-bound library component is amplified
by the PCR to create a new library to be used in the next round. Aptamers are
continuously developed through this on-going process, and their characteristics are
identified using various biological assays.

10. Conventional SELEX technique

11. b. Nitrocellulose Membrane Filtration-Based SELEX
• A nitrocellulose membrane is often used to immobilize proteins in Western blots and
atomic force microscopy (AFM) because it provides simple and rapid protein
immobilization by its non-specific affinity for amino acids.
• In 1968, a method using nitrocellulose membranes was employed by Kramlova’s group
to easily and quickly separate a protein from RNA molecules.
• Because the targets were primarily proteins in the early stage of SELEX, the use of a
nitrocellulose membrane was applied during the separation step. However, these
membranes have some limitations, such as being incapable of binding small molecules
and peptides, and they generally require at least 12 selection rounds.

13. C.AFFINITY CHROMATOGRAPHY BASED SELEX
Affinity chromatography is a method for separating the components from a
biochemical mixture. It is primarily used for the purification of recombinant proteins,
based on a highly specific biological interaction, such as that between a receptor and a
ligand, or an antigen and an antibody.
The immobile phase is generally composed of agarose-based beads, and the beads are
packed onto a column for the washing and elution processes.
In the binding and separation steps of SELEX, affinity chromatography assists in the
selection of only the library components with an affinity for the target, by immobilizing
target molecules on the beads.
In the case of immobilization of proteins, various tags such as glutathione S-transferase
(GST) and the His-tag are utilized and, in the case of small organic molecules, the
targets are covalently fixed on the beads via a chemical reaction, such as coupling with
EDC (1-Ethyl-3-(3 Dimethylaminopropyl) carbo-di-imide) . Thus, SELEX for small
molecules, as well as proteins, can be carried out using this method. Several aptamers
were selected using this method by making an affinity column containing target-
immobilized beads.

14. AFFINITY CHROMATOGRAPHY BASED SELEX

15. D. MAGNETIC BEADS BASED SELEX
Magnetic beads are also used to immobilize the target via an interaction or a
chemical reaction between an affinity tag and the substrate on the beads.
The use of magnetic beads is an especially powerful tool for easy and rapid
isolation of target-immobilized beads with a magnet.
aptamer selection has been involved in linking this magnetic bead-based strategy
to the SELEX technique. Particularly because the library bound to the target is
easily and simply separated from the unbound one by a magnet, many attempts
at selecting target specific aptamer have followed

16. MAGNETIC BEADS BASED SELEX

17. E. Capillary Electrophoresis-Based SELEX
Capillary electrophoresis (CE) possesses several appealing advantages over the
other analytical separation methods in the aspects of speed, resolution, capacity,
and minimal sample dilution.
This method can separate ionic species by their charge, frictional forces, and
hydrodynamic radius under the influence of an electric field.
With this method, an aptamer can be selected by a mobility shift among the
mixture of a target, the library, and the target-library complex.
In particular, the greatest virtue of applying this method to SELEX is that the
successful selection of the aptamer can be achieved within very few rounds,
generally 2–4 rounds, compared to other methods.

18. Capillary Electrophoresis-Based SELEX

19. f. Microfluidic-Based SELEX
In order to select an aptamer more effectively, SELEX using a
microfluidic or chip system was developed.
Since this method is mainly processed on a chip, it is able to enhance
selection efficiency on a small scale.
For example, the DNA aptamer-specific bound to neurotoxin type B
was obtained after a single round of selection using the Continuous-
flow Magnetic Activated Chip-based Separation (CMACS) device
designed by Soh’s group.
Microfluidic technology-based SELEX is catching on as an advanced
method to select aptamers rapidly and automatically.

20. Microfluidic-Based SELEX

21. G. Cell-SELEX
Cell-SELEX is aimed at searching for an aptamer against a whole cell, whereas the
primary targets of other SELEX methods are single highly-purified proteins.
In other words, the targets of Cell-SELEX are extracellular proteins on the cell
surface or unique structures of the cell. In the majority of cases, Cell-SELEX
processes have washing (for adhesive cells) or centrifugation (for suspension cells)
steps during the separation of aptamers, because target immobilization is not
practicable in the solid phase.
In addition, counter selections are necessary in each round to avoid selection of
aptamers that non-specifically recognize the cell surface and that are commonly
located at the surfaces of many cells.
Therefore, this method is complicated, because of the impossibility of immobilizing
targets, and due to counter selection. However, the resulting aptamers, once
selected, are powerful for cell-specific diagnosis, cell-targeted drug delivery, and
cell-specific therapy.

22. Cell-SELEX

23. h. Other Method-Based SELEX
Several methods, such as Atomic Force Microscopy (AFM),
electrophoretic mobility shift assays (EMSA), and surface plasmon
resonance (SPR), have been performed in connection with SELEX.
Although these strategies have the advantage of reducing the
number of selection rounds, the effectiveness of these methods in
selecting the aptamer has not been clearly demonstrated.

24. Advantages of Aptamers
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.

25. Disadvantages of Aptamers
Lower levels of affinities than antibodies.
Aptamers will not bind to some target molecules.
Aptamers identification is expensive and labour intensive.

26. COMPARISON OF APTAMERS AND ANTIBODY
APTAMER ANTIBODIES
Aptamer are oligonucleotide and protein Antibodies are protein in nature
Uniform activity regardless of batch varies from batch to batch.
Investigator determines target site of protein Immune system determines target site of protein.
Wide variety of chemical modifications to molecule
for diverse functions
Limited modifications of molecule
No evidence of immunogenicity. Significant immunogenicity
They are more stable at high temperature and they
can be regenerated easily after denaturation.
Temperature sensitive
Entire selection is a chemical process carried out in
vitro and can therefore target any protein .
Selection requires a biological system, therefore
difficult to raise antibodies to toxins (not tolerated
by animal) or non-immunogenic target.
Aptamers are single stranded DNA or RNA
oligonucleotide or peptides.
Antibodies are monoclonal or polyclonal.

27. Applications of aptamers
1. Aptamers: as drug therapeutics:
 Due to specific and tight affinity to target molecules, and low or no
immunogenicity and toxicity, aptamers are expected to be effective
therapeutics reagents.
 In 2004, the approval by the Food and Drug Administration (FDA) of
Macugen, a vascular endothelial growth factor (VEGF)-specific aptamer,
for the treatment of neovascular (wet) age-related macular degeneration
(AMD), is a prominent landmark in the application of aptamers.
 This drug is a pegylated aptamer, a single strand of nucleic acid with
specificity to VEGF165, which plays a critical role in angiogenesis and
permeability

28. 2. APTAMER: as DRUG DELIVERY SYSTEM:
 Aptamers that bind to internalized cell surface receptors have been exploited to deliver drugs
and a variety of other cargo into cells.
 For example:
 (a)The prostate-specific membrane antigen (PSMA) is an important prostate cancer marker. The
dual aptamer probe—an A10 aptamer for PSMA(+) prostate cancer cells, and a DUP-1 aptamer
for PSMA(−) prostate cancer cells—was invented, and a drug loaded dual aptamer complex was
constructed by loading doxorubicin, an anticancer drug, onto the A10 aptamer strand. As a
result, the doxorubicin can be effectively introduced into the prostate cancer cells.
 (b) Design of an siRNA-aptamer conjugate via a modular streptavidin bridge using an antiPSMA
aptamer for prostate cancer cells (LNCaP).
Applications of aptamers

29. 3. Aptamer: uses in Bio-Imaging:
 Another application is bio-imaging, using an aptamer that is conjugated to
a fluorophore, or other materials such as gadolinium, which is useful for
magnetic resonance imaging (MRI).
 Using aptamers as imaging agents has the advantage of their being non-
toxic, because oligonucleotide moieties are present in the human body.
 As aptamers have high specificity for their target, accurate targeting, and
rapid diffusion through the blood circulation, use of these molecules can
increase the certainty of the results obtained during diagnosis or clinical
analysis.
 Based on these advantages, aptamers have been studied as imaging agents
for cell imaging as well as single-protein imaging.
Applications of aptamers

30. 4. Aptamers: Ease in Western Blot Analysis:
 A Western blot analysis is an analytical technique routinely used to quantify
specific proteins. The procedure includes complicated and elaborate steps and
requires many reagents, such as two types of antibodies.
 a new aptamer-based Western blot strategy that has reduced the procedure to
one step, and easily detects the target protein using only one aptamer.
 Instead of two types of antibodies, the QD-conjugated RNA aptamer specific for
the His-tag (Polyhistidine tag) was employed. This method has the advantages of
requiring less time, not requiring antibodies or 32P, and introducing the
possibility of multiplexing detection.
Applications of aptamers

31. 5. APTAMERS AS DIAGNOSTIC TOOLS:
 As Aptamers are high affinity and specificity, small size, little immunogenicity,
stable structures and ease of synthesis it can be used as diagnostic tools.
 Aptamer based detection assays are expected to detect low concentration
pathogens than conventional antibody based detection assay such as ELISA.
TYPE OF APTAMER VIRUS SPECIFIC TARGET MODE OF ACTION
2’-Fluropyrimidine
containing RNA
Aptamer
HIV-1 Strain R5 Glycoprotein 120 Inhibit cell to cell
movement and virus to
cell infection
Peptide Aptamer C1-1 Hepatitis B virus Viral coat Protein Inhibit viral replication
by blocking capsid
formation.
Applications of aptamers

33. 1. Kyung-Mi Song, Seonghwan Lee and Changill Ban; Aptamers and
Their Biological Applications; Sensors 2012, 12, 612-631;
doi:10.3390/s120100612
2. 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.
3. 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