Aptamers

1. Aptamers as Diagnostic and
Therapeutic agent
Dr. Rakhi Gangil
Doctoral Seminar

2. Introduction
Why aptamers matters?
Aptamer vs Antibody
Types of Aptamers
History
SELEX
Application
Aptamer as Biosensors
Aptamers against pathogens
Advantages and Disadvantages
Conclusion
Outlines

3. Introduction
 (apto: “to fit”& mer: “smallest unit of repeating structure”).
 Single stranded folded oligonucleotides and peptide
 Structure-based drug design strategies
 Three-dimensional structures – well bind to a wide variety of targets from single
molecules to complex target mixtures or whole organisms
size - 20 to 80 bases (6-
26 kDa).

4. Why they matter ??
 Short DNA, RNA, or peptide structures
 Synthesized against small molecules toxins, proteins of viruses , bacteria and even
whole cell
 ​unlimited spectrum of molecular targets​, unbound by limitations of immunogenicity.
 Bind with high specificity and affinity
 Used as synthetic antibodies and similar to monoclonal antibodies
 enormous therapeutic potential for which antibodies are not well suited
 Precise control over syntheis, purity and pharmokinetic profile
 Cutting edge therapeutic tools to treat disease into the 21st century.

5. History
 In 1980s – Concept of joining nucleic acids with proteins
(HIV and adenovirus )
small structured RNAs can bind to cellular proteins with
high affinity and specificity
In 1990 - Larry Gold and Craig Tuerk, developed the SELEX process
– Two sequences isolated from a randomized pool of around
65,500 nucleotide sequences
Nucleic acids could be used to recognize proteins.
Andy Ellington coined the term "aptamer."

6. Aptamer vs Antibodies

7. Types of Aptamers
RNA APTAMER DNA APTAMER PEPTIDE APTAMER
Complex secondary and
tertiary structure
Complex secondary and
tertiary structure
Structure constrains by
scaffold
Diverse 3d structure Less diverse 3d structure
than RNA aptamer
3d structure constraints by
scaffold
Bind target with the entire sequence bind target variable region
only
Require reverse
transcription in SELEX
Inherently more
stable, cheaper,
and easier to produce
-
Biosensor, Diagnostic, Therapeutic applications

8. SELEX
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
(Ellington, A.D & Szostak, J.W., 1990)
Systematic Evolution of Ligands by Exponential Enrichment

9. Random DNA Oligonucleotide Library
(Stoltenburg et al., 2007)

10. A general schemes of SELEX method for DNA and
RNA libraries

11. Steps of SELEX
 Selection
 Amplification
 Conditioning
 Post SELEX modifications

12. Selection
 Immobilization of the target molecule
on a particular matrix
 Affinity chromatography - Column
material like sepharose or agarose
(conventional method)
 Magnetic beads

13. Assessment of SELEX procedure
Quantification of the enriched target-binding
oligonucleotides as well as the amount of non-binding
oligonucleotides
Radioactive markers – drawback cost effective
(Beinoraviciute-Kellner et al., 2005)
Fluorescence labels used for quantification
(Stoltenburg et al., 2005)

14. Amplification
 Only few functional oligonucleotides in result of the selection step
 RNA aptamers
SS RNA RT-PCR cDNA PCR
 DNA aptamers
 PCR with special primers

15. Conditioning
In vitro transcription (RNA aptamers)
ds DNA RNA oligonucleotide
Purification of single strandard DNA (DNA aptamers)
Single strand separation has to be carried out by using streptavidin/biotin
 dsDNA (only one strand biotinylated) bind to streptavidin surfaces (beads or plates)
and separate the strands after DNA denaturation
(Naimuddin et al., 2007)
Asymmetric PCR which uses only one or a much bigger amount of one primer to obtain
ssDNA products (Wu and Curran, 1999)
Transcription with T7 RNA polymerase

16. Post SELEX modifications
 RNA aptamers larger variety of secondary structures
 more sensitive to cellular nucleases
 DNA aptamers - more stable and their biological stability can also be improved.
(Breaker, 1997) (Keefe & Cload, 2008).
 Protective chemical modifications becomes necessary.
 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

17. Nucleic acid modifications

18. Applications
(Breaker, 1997),

19. Aptamer as biosensor
 Analytical device which converts the
Biological response Electrical signals
 Fast and efficient diagnosis of diseases.

20. 20
A schematically represented biosensor
Transducer RecorderReceptor
Antibody
Enzyme
Aptamer etc.
Bioreceptor Biomarker Protein
DNA
RNA etc,

21. Why Biosensors important…?
 Small in size
 Fast in measurement
 Preliminary sample preparation are not required
 Target specific
 Electronic device
 high sensitivity and specificity of biomarker detection

22. Aptamer-Based Sensor
A biosensor that is based on aptamers – Aptasensor
Electrochemical Aptasensor,
Fluorescence-Based Optical Aptasensor
Colorimetric-Based Optical Aptasensor

23. Electrochemical aptasensor
Hybridized
aptamer with
complementary DNA,
immobilized on the gold
surface.
Aptamer bind with
the target
and decrease the
amount of the
aptamer on the
electrode surface
(a) Electrochemical aptasensor using Fe(CN)6 4−/3−
(b) Electrochemical aptasensor using MB
Aptamer folds into the target-binding three-way
junction, altering the electron transfer (eT) and
increasing the observed reduction peak
(c) electrochemical aptasensor using Fc
aptamer folds into the hairpin structure, results in
increased efficiency of eT between the Fc probe and the
electrode surface
(Song et al 2012)

24. Fluorescence-Based Optical Aptasensor
a) Quenching aptamer beacon
b) Assembly aptamer beacon
c) Disassembly aptamer beacon
(Stojanovic et al 2001)

25. Colorimetric-Based Optical Aptasensor
AuNPs or several polymers cause color change - optical detection
technique (colorimetry)
Highly negatively-charged ssDNA
(complementary strand of the aptamer)
Aptamer with targetColor change
(Zhao et al 2006)

26. Aptamer Based ELISA- ALISA
(a) the ELISA method (b) the ALISA method.
An aptamer-linked immobilized sorbent assay (ALISA)
(Vivekananda et al 2006)

27. Aptamer based Rapid diagnostic test
(Lateral flow assay)
A schematic illustration of an AuNPs-based strip assay.
(Xu et al., 2009)

28. Aptamer based western blot analysis
(a) The conventional Western blot analysis (b) The aptamer-based Western blot analysis.
Conjugated RNA aptamer
(Shin et al 2010)

29. Aptamers against Pathogens

30. Avian influenza virus (AIV)
 Hemagglutinin (HA) - a surface glycoprotein
 RNA aptamers P30-10-16 and A-20, that specifically bind the type A
and B of HA, respectively
 Able to distinguish influenza type A from type B or even closely related
strains of the same influenza subtype
Binding affinity more than 15-fold higher than anti-HA monoclonal
antibody
(Gopinath et al.)
Method Virus
Isolation
ELISA RT-PCR qRT-PCR SPR Aptasensor
Detection time 120-170 h 3 h 5 h 3 h 1.5 h
Comparison of Avian Influenza Virus detection time
with different time diagnostic methods

31. Aptamers against Human Immunodeficiency Virus (HIV)
 Aptamers that recognize HIV proteins i.e. Tat and Rev, have
been mainly used in several biosensor systems
 Tombelli et al. generated biosensors (high sensitivity and specificity)
 Surface Plasmon Resonance (SPR)
 quartz crystal microbalance (QCM) techniques.
 They introduced aptamers capable of detecting the HIV-1 Tat
protein, using biotin-streptavidin interaction.
(Tombelli et al 2005)

32. Aptamers against BRSV
 Aptamer based sensor for detection of target mRNA from RSV.
 Oligonucleotide hybridization probes (hairpin shaped molecules) with
a streptavidin binding aptamer sequence was blocked for this assay.
 sensor sensitivity - detect 13 pmol of RSV mRNA in a 4 μL total
volume of a complex biological media.
(Cai et al..,2013)

33. Aptamers and aptamer-based biosensors in viral diagnostics
Virus Aptamer Type Target Detection technique
Influenza
H5N1
RFA006
RHA0385
DNA Haemagglutinin
Surface Protein
Sandwich ELISA
QCM based biosensor
HIV-1 n/d RNA Tat protein
FET based biosensor
QCM based biosensor
SPR based biosensor
HCV E2-B
E2-D
DNA E2 glycoprotein ALISA
Vaccinia PP3
TV01
DNA hemagglutinin
surface protein
fluorescence microscope using Alexa
Fluor 594-labeled aptamer PP3
flow cytometry assay using Cy5-
labeled aptamer
HPV 13, 14,20,28 DNA Epitope of surface
protein of non infected
cell
Confocal Microscope
Chikungunya,
Dengue,
West Nile
spectrum of selected
aptamers
DNA viral envelope proteins
lateral flow chromatographic test strip
fluorescent aptamer-magnetic bead
sandwich assay
Dengue apt_EcoRI n/d EcoRI enzyme—one of
biosensor modules
modular biosensor detecting the
genetic sequences of Dengue genome
( Wandtke et al., 2015)

34. Strategies of antiviral therapy with use of
aptamers
 Blocking of Viral Fusion with the
Target Cell
 Inhibition of Proteins and
Enzymes of Viral Replication Cycle
 Inhibition of Nucleic Acid
Sequences Essential for Virus
Replication Cycle
 Delivery of Therapeutic Molecules
to infected Cells

35. Blocking of Viral Fusion with the Target Cell
 HIV-1 glycoprotein gp120 ligand of T helper cell receptar (CD4) –
RNA aptamer B40 and B40t77 inhibit the binding of gp120 to receptor
(Dey et al, 2005)
 HCV E2 glycoprotein coreceptor for CD81 –
DNA aptamer ZE2 block the E2 glycoprotein (Chen et al 2009)
 Conserved-HA region of Influenza virus –
DNA aptamer A22 bound the HA specific site responsible for
ligation of cell receptor (Jeon et al
2004)

36. Virus Aptamer Type Target Aptamer application method Inhibitory effect
Influenz
a H5N1
A22 DNA HA Balb/c mice intranasaly inoculated
with A22 solution
>90 % decrease in viral
loads in mice
H9N2 HA12-16 RNA gHA1 MDCK infected culture cells
incubated with aptamer
Efficient suppression of
infection of the cells
HIV B-40, B40t77 RNA Gp120-
ccR5
PBMC culture cells inoculated with
aptamer before infection
Inhibition of viral
infectivity
Ch A-1 (Anti-
gp120
aptamer-
siRNA
chimera)
RNA gp120
(aptamer)
tat/rev (si-
RNA)
RAG-Hu mice were injected with
chimera solution
Reduction in tat/rev
mRNA transcript level in
mice 75%-90%
HCV B2 DNA NSB5 Aptamer added to HCV-NS5B in
vitro reaction
Inhibition of polymerase
activity
HSV Aptamer-1 RNA gpD Virus particle preincubated with
aptamer used in VERO cells
Blockade of viral entry
HBV S9 RNA P Protein HepG2.2.15 cells transfected with
plasmid encoding aptamer
Reduction of replicative
intermediates by 80%
Ebola IGB-14 RNA eVP35 Inhibition of EBOV
polymerase activity and
VP36 nucleoprotein
reaction
Aptamers application: in vitro therapeutic experiments and models in vivo.
( Wandtke et al., 2015)

37. Aptamer-siRNA chimeras
 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

38. Antibacterial aptamers
Certain components of the bacteria as well as whole bacterial cells can be
used as ligands for aptamer selection.
S. enterica serovar Typhi
 RNA aptamer S-PS8.4, which specifically binds type IVB pili
 2.0 mg of S-PS8.4 provided 71% inhibition of the invasion of pili
S. Typhi into human monocytes (THP-1 suspended cell line) due to
specific aptamer binding. (Therapeutic application)

39. Continue….
 The aptamer S-PS8.4 covalently immobilized on the surface of
carboxylated single-walled carbon nanotubes – Used in detection
system (aptasensor) (Zelada-Guille´n et al., 2009)
 Highly sensitive - detection of a single CFU of target S. enterica in
an assay that was close to real-time.
 Highly specific - as no signal was detected from Escherichia coli
or Lactobacillus containing samples.

40. Staphylococcus aureus
 62 nt DNA aptamers SA17 and SA61 obtained using whole bacterial
cells as SELEX targets, where every selection round included a negative
selection stage against S. epidermidis.
 Immobilization of these aptamers on the surface of gold nanoparticles
were used for development of detection system
Chang et al. (2013)

41. Continue….
 DNA aptamers capable of recognizing the alfa-toxin of S.
aureus were selected.
 Aptamers AR-27, AR-33, AR-36, and AR-49 significantly
inhibited a-toxin-mediated cell death in Jurkat T cells. cell
survival rate – 50-60% to 80-90% (Therapeutic drug)
(Vivekananda et al., 2014)
 Enterotoxin B of S. aureus was also used as a ligand for the
selection – Aptamer APTSEB1 (used as aptasensor)
(DeGrasse, 2012).

42. E. coli
 E. coli O157:H7 strain can cause a wide range of diseases
 Aptamers against E. coli, both whole bacteria and bacterial
components
 A DNA aptamer against lipopolysaccharide of E. coli O111:B4
Aptamer conjugate with C1qrs protein Antibacterial activity
(Bruno et al., 2008b)
 DNA aptamer targeting outer membrane proteins of an E. coli
Crooks strain for the specific FRET detection of bacteria
(Bruno et al., 2010).

43. Continue…
 DNA aptamer against fimbriae protein of E.coli K88 strain
Li et al. (2011).
 Enteropathogenic E. coli strain K88 was also used for cell-SELEX
of a DNA aptamer. A sandwich-type fluorescent assay was
developed for the detection of E. coli K88 in pure cultures as
well as in spiked biological samples.
(Peng, 2014)

44. Mycobacterium tuberculosis
Tuberculosis widespread dangerous infection - drug-resistant MTB strains.
DNA Aptamer NK2 - whole living bacteria as SELEX target
NK2 aptamer - inhibit the invasion of MTB into macrophages
0.8µg NK2 aptamer – significantly decrease in mycobacteria
in MTB infected mice
(Chen et al., 2012, 2013)

45. Continue..
 Aptamer G9 - bacterial enzyme polyphosphate kinase 2 (PPK2)
 PPK2- synthesis of mycolic acid and other surface
polysaccharides
 High binding affinity and inhibited the activity of the enzyme up
to 50% (therapeutic use)
Shum et al. (2011).

46. Continue….
 Aptamer as MTB detection system
 A sandwich-type system - detection of MPT64 antigen
which is expressed during an active bacterial cell division
 L64QA - Immobilized to capture the analyte molecule
 M64RA - Reporter component.
(Qin et al., 2009)

47. Target Aptamer
type
Application
Salmonella
Pilin structural protein of
S. Typhi
Mixture of OMPs of
S. typhimurium
S. typhimurium whole
live cells
RNA
DNA
DNA
Inhibition of the invasion of pil+ S. Typhi into human monocytes
Potentiometric aptasensor based on single-walled carbon nanotubes.
LOD is 0.2 CFU/ml in bacterial suspension.
Aptamers, immobilized on magnetic beads, were applied for the
capturing of the pathogen in biological samples with subsequent PCR
detection. LOD is 10 CFU/g in biological samples.
Sandwich-type system with fluorescent detection.
LOD is 25 CFU/ml in bacterial suspension.
S. aureus
Whole live cells
alpha-Toxin
DNA
DNA
Single cell detection in bacterial suspension.
Inhibition of a-toxin-mediated cell death in Jurkat T cells
Escherichia coli
OMPs of Crooks strain
Fimbriae protein of K88
strain
DNA
DNA
FRET detection in bacterial suspension
Sandwich-type fluorescent assay
LOD is 2.1103 CFU/ml in biological samples
M. tuberculosis
Whole live cells
MPT64 protein
CFP-10.ESAT-6 protein
DNA
DNA
DNA
Decrease of the amount of mycobacteria in MTB-infected mice,
Sandwich-type system
Detection in clinical samples
Antibacterial aptamers and their application
(Davydova et al., 2015)

48. Advantages
 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
 Can usually be reversible if denatured
 Dyes or functional groups can be readily introduced during
synthesis

49. Disadvantage
 Pharmacokinetic and other systemic properties are variable and
often hard to predict
 Small size makes them susceptible to renal filtration - shorter
half-life
 Unmodified aptamers are highly susceptible to serum
degradation

50. Strategies to Overcome aptamer limitations
 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)

51. Conclusions and future perspective
 Aptamers provide opportunities for structure-based drug design strategies
relevant to therapeutic intervention
 Recent advances in the chemical modifications of nucleic acid suggest
stability,
 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 livestock pathogens increasing repertoire of targets,
 Aptamers are poised to successfully compete with monoclonal Abs in
diagnosis, therapeutics and drug development within the next few
decades

52. Thank
You....