The document provides an extensive overview of aptamers, highlighting their characteristics, selection methods, and various applications in drug delivery and diagnostics. Aptamers are oligonucleotide or peptide molecules with high specificity and affinity for target molecules, developed through a process called systematic evolution of ligands by exponential enrichment (SELEX). The document also compares aptamers to traditional antibodies and discusses their potential in therapeutic interventions and bio-imaging.

1. APTAMER BASED DRUG DELIVERY
PRESENTED BY :- ANURAG PANDEY (M.PHARM)
DEPARTMENT OF PHARMACEUTICS, INSTITUTE OF PHARMACY,
NIRMA UNIVERSITY, AHEMDABAD
UNDER THE GUIDANCE OF DR. TEJAL MEHTA (HOD)
DEPARTMENT OF PHARMACEUTICS, INSTITUTE OF PHARMACY,
NIRMA UNIVERSITY, AHEMDABAD

2. INTRODUCTION
In, Latin aptus/apto – to fit,
and
In, Greek meros – part (Smallest Unit Of Repeating Structure)
ABOUT
 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).

3. IDEAL CHARACTERISTICS
OF APTAMERS
 First, most aptamers bind to targets with high affinity, demonstrating
typical dissociation constants in the pico- to nanomolar range.
Binding sites for aptamers include clefts and grooves of target
molecules (including enzymes) resulting in antagonistic activity very
similar to many currently available pharmaceutical agents.
 Second, aptamers are structurally stable across a wide range of
temperature and storage conditions, maintaining the ability to form
their unique tertiary structures.
 Third, aptamers can be chemically synthesized, in contrast to the
expensive and work-intensive biological systems needed to produce
monoclonal antibodies.

4. APTAMERS TARGET BINDING MECHANISM

5. SYSTEMATIC EVOLUTION OF LIGANDS BY
EXPONENTIAL ENRICHMENT
 Aptamers are typically generated by an iterative screening
process of complex nucleic acid libraries (>1014 shapes per
library) employing a process termed Systematic Evolution of
Ligands by Exponential Enrichment (SELEX).
 SELEX produces ssRNA with specific binding target.
 This is an iterative process of binding, partitioning,
amplifying novel nucleic acids & regeneration, the pool
becomes enriched for ligands that bind the target protein with
high affinity and specificity

6. CONT….
 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’ & 3’ end.
 The pool of DNA is transcribed into an RNA pool & it is
subsequently exposed to the target ligand of interest.
 Affinity column chromatography removes unbound
sequences & identifies the strongest binding sequences &
bound are eluted & amplified by RT-PCR.

7. (SELEX IN SIMPLE)

8. (SELEX IN DETAIL)

9. APTAMER SELECTION METHOD
BASED ON SELEX
1. NITROCELLULOSE MEMBRANE FILTRATION - BASED SELEX
2. AFFINITY CHROMATOGRAPHY & MAGNETIC BEAD - BASED
SELEX
3. CAPILLARY ELECTROPHORESIS-BASED SELEX
4. MICROFLUIDIC-BASED SELEX
5. CELL-SELEX

10. CLASSIFICATION
OF
APTAMERS BASED ON STRUCTURE
APTAMERS
NUCLEIC
ACID
RNA
DNA
XNA
AMINO
ACID
PEPTIDES
AFFIMER

11. COMPARISON OF RNA, DNA, & PEPTIDE APTAMERS
RNA APTAMERS DNA APTAMERS PEPTIDE APTAMERS
COMPLEX SECONDARY &
TERTIARY STRUCTURE
COMPLEX SECONDARY &
TERTIARY STRUCTURE
STRUCTURE CONSTRAINS BY
SCAFFOLD
DIVERSE 3D STRUCTURE LESS DIVERSE 3D STRUCTURE
THAN RNA APTAMERS
3D STRUCTURE CONSTRAINTS
BY SCAFFOLD
BIND TARGET WITH THE
ENTIRE SEQUENCE
BIND TARGET WITH THE
ENTIRE SEQUENCE
BIND TARGET’S VARIABLE
REGION ONLY
BIOSENSOR, DIAGONOSTIC,
THERAPEUTICS APPLICATION
BIOSENSOR, DIAGONOSTIC,
THERAPEUTICS APPLICATION
BIOSENSOR, DIAGONOSTIC,
THERAPEUTICS APPLICATION
AFFIMERS :- The Affimer protein, an evolution of peptide aptamers, is a small, highly stable protein
engineered to display peptide loops which provides a high affinity binding surface for a specific
target protein. It is a protein of low molecular weight, 12–14 kDa, derived from the cysteine protease
inhibitor family of cystatins.

12. COMPARISON BETWEEN
DNA & RNAAPTAMERS
 In contrast to antisense oligonucleotides and small interfering RNAs (siRNAs)
that inhibit translation of proteins by Watson-Crick base-pairing to their
respective messenger RNAs, aptamers bind to existing proteins (and, less
commonly, non-protein targets) with high affinity and specificity, analogous to
monoclonal antibodies.
 RNA and DNA aptamers both have theoretical advantages and proponents, but
aptamers of comparable affinity and specificity can be generated from RNA or
DNA. Since nuclease resistance is critical for aptamer stability in biological
fluids, RNA libraries employed in SELEX are front-loaded with 2'-modified
nucleotides, most commonly 2'-fluoro- or 2'-O-methyl pyrimidines.

13. CONT…
 Probably the biggest current advantage of DNA over RNA aptamers is the
significantly lower cost of chemical synthesis for unmodified DNA
oligonucleotides. However, RNA is preferred by many groups due to the
theoretically higher affinity of RNA aptamers for their target proteins as well as
the greater plasma stability of modified RNA than unmodified DNA.
 Stimulation of the immune system via Toll-like Receptors by double-stranded
regions within RNA aptamers is a valid concern, but modified (“artificial”)
nucleotides do not appear to be potent stimulators of this innate immune
response.

14. CONT…
 Most SELEX libraries have random regions ranging from 20 to 60 nucleotides
(nts), flanked by constant regions for amplification and transcription, and therefore
total lengths ranging from 70 to greater than 100 nts. Sufficient quantities of any
length aptamer can be generated by in vitro transcription for testing in vitro and in
certain small scale animal models.
 However, chemical synthesis is necessary for larger scale applications. Since the
efficiency of chemical synthesis decreases with oligonucleotide length, it is often
necessary to “minimize” or “truncate” an aptamer prior to study in vivo. Although
the technology continues to improve, aptamers longer than 60 nts are not
currently amenable to cost-effective chemical synthesis, and the shorter, the
better.
 Due to the relatively small size (8 kDa to 15 kDa) of truncated aptamers, their
circulating half-lives are limited not by plasma stability but by renal clearance,
which can be improved by conjugation to high molecular weight groups such
as polyethylene glycol (PEG)

15. CELL SURFACE PROTEINS APTAMERS
& THEIR APPLICATION
Receptor Name RNA/
DNA
Selection Technique Delivery Application
Tenascin-C (TN-C) RNA Purified TN-C In vivo tumor imaging
Nucleolin DNA Not applicable Photodynamic therapy (PDT), tumor imaging
Prostate Specific
Membrane Antigen
(PSMA)
RNA Purified extracellular
domain
of PSMA
siRNA delivery, cytotoxin delivery,
Chemotherapeutic drug delivery and
cellular imaging
gp120 RNA Purified recombinant
gp120
siRNA delivery
Transferrin receptor
(TfR)
RNA/
DNA
Purified extracellular
domain of mouse TfR
Protein targeting to lysosome

16. CONT…
Receptor Name RNA/
DNA
Selection Technique Delivery Application
Mucin-1 (MUC-1) DNA Recombinant peptides Photodynamic therapy (PDT),
Radionuclide delivery
Protein tyrosine
kinase-
7 (PTK7)
DNA Cell SELEX using T-cell
acute lymphoblastic
leukemia
(ALL) cell line
Chemotherapeutic drug delivery
Immunoglobin heavy
mu chain (IGHM)
DNA Cell SELEX using
Burkitt’s
lymphoma cell line
(Ramos)
Micelle nanoparticles for drug delivery
Epidermal growth
factor receptor (EGFR)
RNA Purified extracellular
domain
of EGFR
Nanoparticle delivery

17. COMPARISON
CHARACTERISTICS APTAMERS ANTIBODIES
NATURE OLIGONUCLEOTIDE & PROTEIN PROTEIN
ACTIVITY UNIFORM REGARDLESS OF BATCH VARIES BATCH TO BATCH
CHEMICAL
MODIFICATION
HUGE MODIFICATION CAN BE DONE LIMITED
IMMUNOGENICITY NO EVIDENCE (ABSENT) SIGNIFICANT
EFFECT OF TEMP. STABLE AT HIGH TEMP. & CAN BE
REGENERATED AFTER DENATURATION
TEMP. SENESITIVE
METHOD OF
PRODUCTION
ENTIRE SELECTION IS IN VITRO
CHEMICAL PROCESS
SELECTION REQUIRES A
BIOLOGICAL SYSTEM, SO
COMPARTIVELY DIFFICULT
STRUCTURE SINGLE/DOUBLE STRANDED DNA OR
RNA OLIGONUCLEOTIDE OR PEPTIDES
THEY ARE MONOCLONAL &
POLYCLONAL
SITE TARGETING INVESTIGATOR DETERMINES IT IMMUNE SYSTEM DETERMINES IT

18. CLASSIFICATION OF APTAMERS
BASED ON DIFFERENT TECHNIQUES
1- APTAMER-SELEX
2- APTAMER-SMALL
MOLECULE CONJUGATED
SYSTEM
3- APTAMER-NANO-PARTICLE
CONJUGATED SYSTEM
A- CYTOTOXIC DRUG B- LINKERS
I- CHEMICAL LABILE
LINKER
II- NON-LABILE LINKER
A- INORGANIC
NANOMATERIALS
B- ORGANIC
NANOMATERIALS
A. ACID CLEAVABLE LINKER
B. DISULFIDE LINKER
A. PEPTIDE LINKER
B. BETA-GLUCORONIDE
LINKER
A. GOLD NP’S
B. NANOSCALE IRON
OXIDES
C. MESOPOROUS SILICA
NP’S
D. QUANTUM DOT’S
A. LIPOSOMES
B. POLY (LACTIC-CO-
GLYCOLIC ACID
C. POLYMERIC MICELLES
D. DENDRIMERS
E. SERUM ALBUMIN NP’S
F. DNA NP’S
NOTE :- NANOPARTICLES (NP’S)

19. CONT…
Aptamer-
Functionalized Targeted
Drug
Delivery Systems
Advantage Disadvantage
Aptamer-small molecule
conjugated systems
Good chemical
stability,
isotropic properties
Low drug loading,
complex and costly
procedures
Aptamer-nanomaterial
conjugated systems
High loading capacity Unpredictable risks,
complex and costly
procedures

20. APPLICATION OF APTAMERS
FOR NEW DRUG
IN, DRUG
DELIVERY
AS,
TEHRAPEUTIC
TOOL
IN, DIAGNOSIS
OF DISEASE
IN, FOOD
INSPECTION
IN, HAZARD
DETECTION
IN, BIO-IMAGING
AS, ANALYTICAL
REAGENT

21. APTAMERS: AS DRUG THERAPEUTICS
TWO MAJOR CONCERN
1. IN 2004, the approval of FDA of Macugen, a vascular endothelial growth
factor (VEGF)-specific aptamer, for the treatment of neovascular (wet) age
related macular degradation (AMD), is a prominent landmark in the application
of aptamers. This drug is pegylated aptamer, a single strand of nucleic acid with
specificity to VEGF165, which plays a critical role in angiogenesis &
permeability.
2. Additionally, regado bioscience has developed REG1 as a new aptamer drug for
anti-coagulation, & this aptamer drug is currently in Phase-II clinical trials.
REG1 (congulation factor IXa-specific aptamer) & RB007 (oligonucleotide
antidote of RB006 aptamer). RB006 is a 2’-ribo purine/ 2’-fluoro pyrimidine
aptamer & is conjugated to a 40kDa PEG to protect the aptamer against
nuclease-mediated degradation.

22. CONT..
Aptamer Molecular Target Associated
Disease
Aptamer Structure
Macugen Vascular
endothelial growth
factor (VEGF)
Age-related macular
degeneration
5′-CGGAAUCAGUGAAUG
CUUAUACAUCCG-3′
AS1411 Nucleoin Cancer 5′-d(GGTGGTGGTGGT
TGTGGTGGTGGTGG)-3′
Sgc8 Protein tyrosine
kinase 7 (PTK-7)
Cancer 5′-ATCTAACTGCTGC
GCCGCCGGGAAAATA
CTGTACGGTTAGA-3′
TD05 Immunoglobulin μ
heavy chains
(IGHM)
Lymphoma 5′-AACACCGGGAGGAT
AGTTCGGTGGCTGTTCA
GGGTCTCCTCCCGGTG-3′
ARC1779 A1 Domain of von
Willebrand factor
(vWF)
Thrombotic
microangiopathies and
carotid artery disease
5′-GCGUGCAGUGCCU
UCGGCCGTGCGGT
GCCUCCGUCACGC-3′
TBA α-Thrombin Thrombosis 5′-GGTTGGTGTGGTTGG-3′

23. APTAMER IN DRUG DELIVERY SYSTEM
 The prostate-specific membrane antigen (PSMA) is an important prostate cancer
marker. The dual aptamer probe-an A10 aptamer for PSMA(+) prostate cancer
cells, & a DUP-1 aptamer for PSMA(-) prostate cancer cells-was invented, & a
drug loaded dual aptamer complex was constructed by loading doxorubicin, an
anti-cancer drug, onto the A10 aptamer strand. As a result, the doxorubicin can be
effectively introduced into the prostate cancer cells.
 Design of an siRNA-aptamer conjugate via a modular streptavidin bridge using an
anti-PSMA aptamer for prostate cancer cells (LNCaP).
SOURCE :-FROM SLIDESHARE APTAMER & ITS APPLICATION BY ACHYUT BORA

24. APTAMERS USE IN BIO-IMAGING
 In this aptamer is conjugated to a fluorophore, or 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 & 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 an imaging agents for
cell imaging as well as single-protein imaging.

25. APTAMERS IN WESTERN BLOT
ANALYSIS
 A western blot analysis is an analytical technique routinely used to quantify
specific protein.
SOURCE :-FROM SLIDESHARE APTAMER & ITS APPLICATION BY ACHYUT BORA

26. USE OF APTAMERS AS
DIAGNOSTIC TOOL
 Aptamer based detection assays are expected to detect low concentration
pathogens than conventional antibody based detection assay such as ELISA.
VIRUS TYPES OF APTAMER TARGET
SPECIFIC
MODE OF ACTION
HIV-1 STRAIN
R5
2’-FLUROPYRIMIDINE
CONTAINING RNA
APTAMER
GLYCOPROTEIN
120
INHIBIT CELL TO
CELL MOVEMENT &
VIRUS TO CELL
INFECTION
HEPATITIS B
VIRUS
PEPTIDE APTAMER C1-1 VIRAL COAT
PROTEIN
INHIBITING VIRUS
REPLICATION BY
BLOCKING CAPSID
FORMATION

27. PRO’S & CON’S OF APTAMERS
PRO’S CON’S
Ability of binding to proteins with high affinity &
specificity.
Pharmacokinetics & other systemic properties are
variable & often hard to predict.
Very small in size but can easily distinguish
between very closely related protein also.
Small size make them susceptible to renal
filtration.
Active at different temperature & pH as well. Unmodified aptamers are highly susceptible to
serum degradation.
Long shelf life. Have shorter half-life.
Low/Non-Immunogenic & non-toxic molecules. Streptavidin was shown to elicit immunogenic
responses-may limit its use as a part of the
delivery vehicle.
Production is done with automated synthesis i.e.,
In-vitro so the cost is low when compared to other
targeting approach.
Aptamer identification is expensive & labour
intensive,

28. CONCLUSION
 Aptamer provides opportunities for structure-based drug design
strategies relevant to therapeutic intervention.
 Recent advances in the chemical modifications of nucleic acids suggest
that one of the major barriers to use is stability & that can be overcome.
 The high affinity & Specificity of aptamers rival with antibodies makes
them a promising tool in diagnostic & therapeutic application.
 We should expect more aptamers to be isolated in the near future against an
ever increasing repertoire of targets, using these different SELEX
approaches with increased speed & efficiency.
 Aptamers are poised to successfully compete with MAB’s in
therapeutics & drug development within the next few decades.

29. REFERENCES
 REVIEW ARTICLES
1. Aptamers for Targeted Drug Delivery, Partha Ray and Rebekah R. White & Department of
Surgery, Duke University Medical Center, DUMC Box 103035, Durham, NC 27710, USA
2. Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery
Systems, Feng Jiang, International Journal of Molecular Sciences ISSN 1422-0067
3. Tumor-Targeted Drug Delivery with Aptamers, Yin Zhang,
 TECHNICAL & INTERNET SOURCES
1. Aptamer Wikipedia.
2. Slide-share powerpoint presentation as well.
3. Rest sources will be mentioned on slide.

30. THANK
YOU
EVERYONE