APTAMERS

BY: SIRISHA.V.MUKKAVALLI
BIOTECHNOLOGY 4/4
07311A2310
 TABLE OF CONTENTS
1.INTRODUCTION

2. PROPERTIES

3. APTAMER TYPES

4. APTAMER PRODUCTION

5. POST- SELEX MODIFICATION

6. APTAMER VS. ANTIBODY

7. APPLICATIONS

8. APTAMERS UNDERGOING PRE-CLINICAL TRIALS

9. ADVANTAGES

10. CONCLUSION
 INTRODUCTION

 15-40 nucleotides long

 Bind tightly to a specific

molecular target

 "aptamer" is derived from

the Latin word aptus,
meaning “to fit",

 It emphasizes lock-and-
key relationship between
aptamers and their binding
partners
 PROPERTIES
 Composition :
-15-40 nucleotides in length
- May be composed of DNA; RNA; or nucleotides with a
chemically modified backbone

 Nature of Interaction :
- Complementary base pairing of these nucleotides generates
secondary structures
-The various combinations of these secondary structures will
result in tertiary structures
-Tertiary structure bind to targets via van der Waals, hyodrgen
bonding and electrostatic interaction
 Selectivity:
-Surface area of interaction between aptamer and
molecular target is relatively large

-Small changes in the target molecule disrupts

aptamer association

- Hence aptamers distinguish between closely related

but non-identical members of a protein family, or
between different functional or conformational
states of the same protein
Validated Therapeutic Targets
APTAMER TYPES

APTAMERS ARE OF 2 MAIN TYPES :

I) RNA Aptamers

II) DNA Aptamers

 APTAMER PRODUCTION
Aptamer production involves the following
steps:-

i. Pool Generation

ii. Selection

iii. Amplification

iv. Aptamer Isolation

1. Pool Generation :
 Pool of nucleic acid molecules are generated using standard
automated oligonucleotide synthesis methods

 Problem: natural RNAs/ DNAs have short half-lives in

serum due to nuclease degradation,

 Solution : Modify nucleotides

Ex:
Add methyl group at 2'-ribose position on the
oligonucleotide backbone.
 2. Selection
 Library of nucleotide sequences are exposed to the target
protein and are incubated for a period of time

 Molecules in the library with weak or no affinity for the

target have a tendency to remain free in solution

 Those with some capacity to bind will tend to associate

with the target.

 The target-bound molecules, among which are the highest

affinity aptamers, are purified away from the target and
used for the subsequent steps in the SELEX process.
3. Amplification
 The captured, purified sequences are "amplified", to
generate a library of molecules enriched with
oligonucleotides that bind to target

 One complete cycle of

Binding
Partitioning
Amplification

is referred to as a "round" of SELEX.

4. Aptamer Isolation
 After 5-15 cycles of SELEX process, the library of
molecules is reduced to a small number that bind tightly
to the target of interest.

 Nucleotide sequences of individual members of the library

are determined.

 The target binding affinity and specificity of selected

sequences are then measured and compared
 Post – SELEX Modifications
Reason for Post-SELEX Modifications :-
 Initial aptamer sequences isolated by SELEX are 70 to 80
nucleotides long.

 Commercializing aptamers of this length would be difficult &

expensive to produce using current manufacturing techniques.

Steps:

i. Minimization: Length is reduced without compromising the

affinity, specificity or functional activity
ii. Optimization: use sequence and chemical modifications to
improve an aptamer’s affinity
iii. Prolonging the time spent in the body: To slow the rate of
excretion from the body, we increase the size of the aptamer by
attaching it to another molecule known as polyethylene glycol
 APTAMERS VS. ANTIBODY

APTAMERS ANTIBODIES

 Denatured aptamers can  Limited shelf life and are

be regenerated in a few prone to degradation
minutes
 Identification of
 Stable during long term antibodies that recognize
storage targets under conditions
other than physiological
 Can be identified through conditions is not feasible.
an in vitro process not
requiring animals  Requires the use of
animals
 Reporter molecules may
be attached to aptamers  Labeling of antibodies can
at precise locations not cause loss in affinity
involve in binding.  May elicit unwanted
 Not good immunogens immune response
 APPLICATIONS
1. Pathway Elucidation : Use aptamers as specific inhibitors

2. Purification : In affinity chromatography

3. Cancer :Binds to certain growth factors that help in tumor

development and hence inactivate them

4. Biosensors: RNA aptamers can be used not only to detect

the presence of a particular analyte as well as concentration
difference
 Aptamer in Pre-Clinical trials
 NX1838:

- It is an aptamer conjugated to PEG

- It is directed against VEGF-165

- It inhibits binding of VEGF to vein endothelial cells

- It is comparable to anti-VEGF monoclonal antibodies

 Advantages
 High specificity & high affinity to the target.

 They are low or non immunogenic & nontoxic molecules.

 Production of aptamers is done in vitro using automated

synthesis.

 Less cost of oligonucleotide synthesis.

 Aptamers can function at different temperatures, pH and

buffers.

 They have an unlimited shelf-life.

 Conclusion
 Most experiments and research were done in vitro.

 To extend aptamers to in vivo applications, it must be

taken into consideration the possible interactions of
aptamers with various biological environments and
tissues

 Hence, although aptamers are not expected to replace

antibodies they will be another weapon that can be used in
the therapeutics
 References
1. “Aptamers- The New Frontier in Drug-Development “Bob Carlson Biotechnology
Healthcare April 2007

2. “Radiolabled aptamers for tumor imaging and therapy” A.C.Perkins Q J Nucl Mol
Med Imaging 2007

3. www.cs.duke.edu

4. www.archemix.com

5.http://bio349.biota.utoronto.ca/20069/bio349jerry1/index.html

6. Rahimi, F, et al. (2010), “RNA aptamers generated against oligomeric Abeta40

recognize common amyloid aptatopes with low specificity but high sensitivity”,
David Geffen School of Medicine, Department of Neurobiology, Los Angeles,
California.

7. www.copewithcytokines.de

8.www.si.mahidol.ac.th/department/Biochemistry/home/content/Chatchawan_eng.ht
m