Aptamers provide 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, stability, can be overcome. The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and 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 and efficiency. Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades.

1. WELCOME

2. Aptamers as TherapeuticsAptamers as Therapeutics
Surender Kumar
P1688

3.  (apto: “to fit”& mer: “smallest unit of repeating
structure”)
 single stranded folded oligonucleotides and peptide that bind to
molecular (protein) targets with high affinity and specificity
 Provide opportunities for structure-based drug design strategies
relevant to therapeutic intervention
 Can be prepared using SELEX
Aptamers combine many of the advantages of oligonucleotides and
antibodies- “Chemical Antibodies”
Based on their three-dimensional structures, Aptamers can well-fittingly bind to a wide
variety of targets from single molecules to complex target mixtures or whole organisms

5. SELEX
 Systematic Evolution 0f Ligands by Exponential Enrichment
 This method, described primarily in 1990
(Ellington, A.D & Szostak, J.W., 1990)
One of the crucial steps of a SELEX process with outstanding importance for the selection of aptamers with high affinity and
specificity is the efficient partitioning between target-binding and non-binding oligonucleotides

6.  Inorganic components
 Zn2+ Ciesiolka et al.
(1995)
 Ni2+ Hofmann et al. (1997)
 Small organic molecules
 Ethanolamine Mann et al. (2005)
 Theophylline Jenison et al. (1994)
 Malachite green Grate and Wilson (2001)
 Amino acids
 L-Arginine Geiger et al. (1996)
 L-Citrulline Famulok
(1994)
 L-Valine Majerfeld and Yarus (1994)
 Carbohydrates
 Cellobiose Yang et al. (1998)
 Chitin Fukusaki et al. (2000)
 Sephadex Srisawat et al. (2001)
 Antibiotics
 Kanamycin A Lato et al. (1995)
 Kanamycin B Kwon et al. (2001)
 Streptomycin Wallace and Schroeder, (1998)
Current opinion in chemical biology

7. Random DNA Oligonucleotide Library
 Starting point of a SELEX
 consists of a multitude of ssDNA fragments
(1015
molecules)
 comprising a central random region of 20–
80 nt flanked by different specific
sequences of 18–21 nt, which function as
primer binding sites in the PCR
 longer random sequence pool may provide
better opportunities for the identification of
aptamers
(Marshall and Ellington, 2000)

8. Selection

9.  quantification of the enriched target-binding oligonucleotides as
well as the amount of non-binding oligonucleotides of each
selection round have to be determined.
 Radioactive markers
(Beinoraviciute-Kellner et al., 2005; Ellington and Szostak, 1990; Shi et al., 2002)
 Fluorescence labels may be used for quantification
(Stoltenburg et al., 2005; Davis et al., 1997; Rhie et al., 2003)

10.  Only few functional oligonucleotides in result of the selection step
 At this stage, SELEX processes for the generation of RNA and DNA
aptamers differ significantly
 RNA oligonucleotides
 firstly have to be passed through a RT-PCR. As a result the
corresponding cDNA is achieved, which is amplified in a subsequent PCR
 ssDNA aptamers
 merely have to be amplified by PCR, where special primers can be used
to provide the aptamers with additional properties

11.  The conditioning step is necessary to prepare the amplified oligonucleotide pool
 After the preceding PCR
 the enriched pool is available as dsDNA.
 A transcription with T7 RNA polymerase has to follow in case of RNA aptamers.
 The resulting RNA molecules are used as input in the following SELEX
round.
 ssDNA aptamers, single strand separation has to be carried out
 use the streptavidin/biotin. ( Fitzwater and Polisky, 1996)
 the dsDNA (only one strand biotinylated) bind to streptavidin surfaces (beads or plates) and separate the
strands after DNA denaturation (Naimuddin et al., 2007)
 perform an asymmetric PCR which uses only one or a much bigger amount of one primer to obtain
ssDNA products (Wu and Curran, 1999)

12. Bacterial plasmids
Sequencing dsDNA pool
(thousands of sequences)
Modified plasmids
Delivery into bacteria
Only bacteria containing an aptamer insert grow.
Each colony contains an individual aptamer sequence.
Bacteria Plasmids from
each colony are sequenced
Sequenced Random site of
aptamer sequence is unravelled

13.  chemically modified oligonucleotide libraries
 with the goal to increase the complexity of a library
 To introduce new features like functional groups providing new possibilities for the
interaction with target molecules
 To enhance the stability of oligonucleotide conformations
 To increase the resistance to nucleases
(Jayasena, 1999; Klussmann, 2006; Kopylov and Spiridonova, 2000; Kusser, 2000)

14. NUCLEIC ACID MODIFICATIONS
Name Description Function
2’ –Aminopyrimidine Substitution at the 2’ position of a pyrimidine with
NH2 group
Increased stability
2’ -O-methylpurine Substitution at the 2’ position in the sugar moiety
with a methoxy group
Cheap, easy to synthesise and
enzymatic incorporation
3’ and 5’
phosphorothioate caps
Replacement of an oxygen atom with a sulphur
atom in the phosphate backbone to form
phosphorothioate linkages and caps
Phosphorothioate linkages are chiral
and form diastereomeric aptamers
thus increasing stability
2’ -Fluoropyrimidine Substitution at the 2’ position of a pyrimidine with
a fluorine molecule
Increased stability
2’ -Azido NTPs Substitution at the 2’ position with N3 Effective cellular internalisation
L-RNA and L-DNA L-Ribose/deoxyribose used instead of usual D-
ribose thus forming mirror image aptamers
Enzymatic degradation of L-
RNA/DNA is lower because of the
lack of nuclease Compatibility
Locked nucleic acids
(LNA)
Bicyclic ring with a furanose ring bridged between
the 2’ -O, 4’-C-methylene bridge
Very high affinity to target sequences
and very high stability

16. Name
(Company)
Composition Target Indication Current
Phase
Refrence
Pegaptanib sodium/
Macugen
(Pfizer/Eyetech)
2′-O-methyl purine/2′-fluoro
pyrimidine with two 2′-ribo
purines conjugated to 40 kDa PEG,
3′ inverted dT
Vascular
endothelial
growth factor
Age-related
macular
degeneration
Approved in
the US and
the EU
Ng et al., 2006
Chakravarthy et
al., 2006
AS1411/ AGRO001
(Antisoma)
G-rich DNA Nucleolin Acute myeloid
leukaemia
Phase II Bates et al., 2009
REG1/RB006 plus
RB007
(Regado Biosciences)
2′-ribo purine/2′-fluoro
pyrimidine (RB006)/40 kDa PEG
plus 2′-O-methyl antidote
(RB007)
Coagulation
factor IXa
Percutaneous
coronary
intervention
Phase II Cooper et al.,
2008
ARC1779
(Archemix)
DNA and 2′-O-methyl with a
single phosphorothioate linkage
conjugated to 20 kDa PEG, 3′
inverted dT
A1 domain of von
Willebrand factor
Thrombotic
microangiopathies
and carotid artery
disease
Phase II Krieg, 2006
NU172
(ARCA biopharma)
Unmodified DNA aptamer Thrombin Cardiopulmonary
bypass to maintain
steady state of
anticoagulation
Phase II Sheehan & Lan,
1998
ARC1905
(Ophthotech)
2′-ribo purine/2′-fluoro
pyrimidine conjugated to 40 kDa
PEG, 3′ inverted dT
Complement
component 5
Age-related
macular
degeneration
Phase I Goebl et al., 2007
NOX-E36
(NOXXON Pharma)
l-RNA with 3′-PEG CCL2 Type 2 diabetes,
diabetic
nephropathy
Phase I Kulkarni et al.,
2009

17. DELIVERY OF CYTOSTATICS
A specific cytotoxic aptamer–doxorubicin (Dox) conjugate. The construct is internalized via endosome,
where the acidic environment favors the cleavage of the bond between the aptamer and Dox molecule.
The antibiotic diffuses through the endosome membrane, penetrates the nucleus and intercalates into
genomic DNA, causing cytotoxic effects.
Bifunctional conjugates
Decreases non-specific
internalization of Dox
greatly enhances its uptake
by target cells
the cytotoxic effect of Dox
was preserved

18.  Data on drug biodistribution
 Monitoring the therapeutic effect in real time
 SPION– Apt–Dox conjugates
 Dual Purpose
 Both detection and elimination of Prostate cancer cells (PCa)
 A10 RNA aptamer specific for PSMA
SPION- Apt Conjugate
Imaged with NMR and displayed high
specificity for PSMA-expressing LNCaP
cells but not for a PMSA-non-expressing
PC-3 cell line
SPION– Apt–Dox conjugates Targeted Delivery

19.  Gold–Silver nanorods (Au–Ag NRs)
 high near-infrared light absorption
 Aptamer Canjugates with Au-Ag NRs
 offers spatial precision in targeted treatment
Huang, Y. F., Sefah, K., Bamrungsap, S., Chang, H. T. and Tan, W. 2008. Selective
photothermal therapy for mixed cancer cells using aptamer-conjugated nanorods. Langmuir;
24(20): 11860–5.
Huang, Y. F., Sefah, K., Bamrungsap, S., Chang, H. T. and Tan, W. 2008. Selective
photothermal therapy for mixed cancer cells using aptamer-conjugated nanorods. Langmuir;
24(20): 11860–5.
Aptamer-directed NRs
 Efficient photothermal
Convectors
 Promoted selective destruction
of the target cells.
 Efficient photothermal
Convectors
 Promoted selective destruction
of the target cells.

20.  aptamer–liposome conjugates
 cargo internalization via
 Fusion of membranes
 Endosome-mediated delivery of liposomes to the
lysosome
Major deficits
1.Stability
2.Nonspecific interactions which were reported
during long-period incubations
Major deficits
1.Stability
2.Nonspecific interactions which were reported
during long-period incubations

21.  Amphipathic unit
 hydrophilic oligonucleotide
 a hydrophobic polymer
 In aqueous solution, self-assembled into a polarized
three-dimensional structure
 Micelle system
 enables delivery of targeted intracellular drugs doped
inside the nanostructure as well as of therapeutic
aptamers into the cell by simple membrane fusion.
Micellization could be also used as a general strategy to
promote binding of low-affinity aptamers

22. Chimeric DNA molecules adapted to simultaneously
recognize two different target proteins
Apt for CD16α Apt for c- MET
coupling with different PEG
moieties and nucleotide linkers
promoted antibody dependent
cellular cytotoxicity
***the bsA17 aptamer, mediated cell lysis with a magnitude similar to cetuximab***

23.  Attractive approach to gene silencing with the aid of aptamers
(Zhou et al., 2008)
Aptamer siRNA chimera targeted at HIV1 gp120 glycoprotein
selectively internalized into HIV-infected cells
Down regulated the tat/rev gene expression
Inhibitory conjugate able to suppress viremia for up to 3 weeks after the final treatment but also
to evade immune response, as no significant elevation in interferon induced genes

24. C. In the presence of the targets the aptamers fold into their three-dimensional
structures, thus opening the nanocage filled with desired molecules
A. DNA nanorobot. A nanoscale cage encapsulating cargo molecules is locked with two
aptamer ‘locks’. Each combines an aptamer towards a chosen target and a complementary
oligonucleotide strand
B . The cage is made of scaffold DNA fastened with multiple DNA staples

25. Aptamer Antibodies
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.
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
Resistant to temperature insult Temperature sensitive

26.  Produced chemically in a readily scalable process
 Not prone to viral or bacterial contamination
 Non-immunogenic
 more efficient entry into biological compartments
 Able to select for and against specific targets and to select against
cell-surface targets
 Can usually be reversibly denatured
 Dyes or functional groups can be readily introduced during
synthesis

27.  Pharmacokinetic and other systemic properties are variable
and often hard to predict
 Small size makes them susceptible to renal filtration
 have a shorter half-life
 Unmodified aptamers are highly susceptible to serum
degradation
 Aptamer technologies are currently largely covered by a
single intellectual property portfolio

28.  Aptamers can be optimized for activity and persistence under physiological conditions
during selection or during structure–activity relationship and medicinal chemistry studies
conducted after discovery
Guo et al., 2008
 Addition of conjugation partners such as polyethylene glycol or cholesterol can increase
circulating half-life
Healy et al., 2004
 Chemical modifications incorporated into the sugars or internucleotide phosphodiester
linkages enhance nuclease resistance
Burmeister et al., 2006

30. Nuclease resistance
• Aptamers composed of unmodified nucleotides have half-lives in the blood that can be as
short as 2 minutes Griffin et al., 1993
• Methods to overcome nuclease Susceptibility
– Modified composition aptamers
• Increase purine residues
– site-specific introduction of nuclease-resistant modifications
• inverting the nucleotide at the 3′-terminus
• changing of the 2′-OH groups of ribose to 2′-F or 2′-NH2 groups or 2′-O-methyl substituted
nucleotides
• A 3′-end capping
– streptavidin-biotin, inverted thymidine (3′-idT, creates a 3′-3′inkage)
• 5′ caps
– amine, phosphate, polyethylene glycol (PEG), cholesterol, fatty acids, proteins, etc.)
(Dougan et al., 2000; Klussmann, 2006; Marro et al., 2005)
• Locked nucleic acids (LNAs)
– the sugar is made bicyclic by covalently bridging the 2′-oxygen and the 4′-carbon with a methylene
group

31.  molecular mass cutoff for the renal
glomerulus is 30–50 kDa
 Aptamers: 5–15 kDa
 Methods to avoid renal filteration
 Conjugation to polymers
 Cholesterol conjugation
 PEG conjugation
 May lead to reduction of activity

32. Pharmacokinetics of aptamers conjugated to different molecular mass pegs.
Pharmacokinetic profiles of 39-mer 2′-deoxy purine, 2′-o-methyl pyrimidine composition
aptamers. these aptamers were unconjugated or conjugated to either 20 kda polyethylene
glycol (peg) or 40 kda peg and administered intravenously to cd-1 mice (n = 3 per time point) at
10 mg per kg. data redrawn from
Fontana, D. J., Epstein, D. E. & Wilson, C. RNA as the drug discovery tool. 1. Aptamer drug
development. 5). Aptamer therapeutics. Idenshi Igaku Mook 4, 61–70 (2006).

33.  Antibodies to oligonucleotide conjugation partners
 Innate immune activation
 TLR3 ds RNA
 TLR7 and TLR8 ss RNA
 TLR9 unmethylated CG motifs(CpG) in DNA
 Anticoagulation
 consequence of low-affinity interactions between the
oligonucleotide and protein components of the clotting cascade
 Complement activation
 inter action of oligonucleotides with complement factor H

34.  Aptamers provide 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, stability, can be
overcome
 The high affinity and specificity of aptamers rival antibodies and
make them a promising tool in diagnostic and 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 and
efficiency
 Aptamers are poised to successfully compete with monoclonal
Abs in therapeutics and drug development within the next few
decades

35.  Keefe, A. D., Pai, S and Ellington, A. 2010. Aptamers as therapeutics.
nature reviews, 9:537-50.
 Huang, Y. F., Sefah, K., Bamrungsap, S., Chang, H. T. and Tan, W.
2008. Selective photothermal therapy for mixed cancer cells using
aptamer-conjugated nanorods. Langmuir; 24(20): 11860–5.
 Stoltenburg, R., Reinemann, C. and Strehlitz, B. SELEX—A
(r)evolutionary method to generate high-affinity nucleic acid ligands
Biomolecular Engineering 24 (2007) 381–403
 Nimjee, S. M., Rusconi, C. P. and Sullenger, B. A. Aptamers: an
emerging class of therapeutics. Annu Rev Med 2005, 56:555-583.