Workshop - Best of Both Worlds_ Combine KG and Vector search for enhanced R...
Víctor M. González - Introducción a la tecnología de aptámeros y posibles aplicaciones terapéuticas
1. Introduction to aptamer technology and possible therapeutic applications
APTÁMEROS: NUEVAS POSIBILIDADES DE DESARROLLO
Y APLICACIONES EN BIOTECNOLOGÍA
17 de Noviembre de 2011
Introducción a la tecnología de aptámeros y
posibles aplicaciones terapéuticas
Víctor M. González
Servicio de Bioquímica-Investigación
Hospital Ramón y Cajal (IRYCIS)
2. Introduction to aptamer technology and possible therapeutic applications
APTAMERS: NEW DEVELOPMENT POSSIBILITIES AND
APPLICATIONS IN BIOTECHNOLOGY
November 17, 2011
Introduction to aptamer technology and
possible therapeutic applications
Víctor M. González
Servicio de Bioquímica-Investigación
Hospital Ramón y Cajal (IRYCIS)
3. Introduction to aptamer technology and possible therapeutic applications
Number of publications related to
aptamers (PubMed)
600
500
Publications
400
300
200
100
0
Year
4. Introduction to aptamer technology and possible therapeutic applications
What is an aptamer?
ssDNA TGATCC ATTCGGATCAAGCTAGC
RNA UGAUCC AUUCGGAUCAAGCUAGC
ATTCGGATCAAGCTAGC
CCTAGT
5. Introduction to aptamer technology and possible therapeutic applications
Secondary structures
Chastain, M. and Tinoco Jr., I., (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 131-177.
6. Introduction to aptamer technology and possible therapeutic applications
Tertiary structure
The folded aptamers adopt
stable conformations that
allow molecular recognition
of the target
Overview of the Vitamin B12
aptamer structure
Nature Structural Biology, 7(1):53-57
7. Introduction to aptamer technology and possible therapeutic applications
Aptamers are obtained…
• …from libraries of oligonucleotides with random
sequences
• …by an in vitro method called Systematic Evolution of
Ligands by Exponential Enrichment (SELEX)
consisting of successive rounds of selection-
amplification
8. Introduction to aptamer technology and possible therapeutic applications
Oligonucleotide library
Random positions
Oligo 1 20-90 N Oligo 2
PCR amplification
Theoretical : N = 40 440 = ~ 1024 different molecules
Real: ~ 1015 different molecules
9. Introduction to aptamer technology and possible therapeutic applications
Libraries used for SELEX
• Classical library
• Libraries with structural constraints
– The variable region is located between conserved sequences that
produce defined structures (hairpin, G-Quartett, pseudoknot)
• Libraries based on known sequences
– It has a known sequence including small amounts (1-30%) of all other
nucleotides allow certain variations along the sequence
• Libraries free of fixed sequences
– Method: tailored SELEX
– Very short conserved sequences to which an adapter is added that is
removed after each PCR
• Libraries based on genomic sequences
– The genomic DNA is fragmented (50-500 nt) and the conserved
sequences are added
10. Introduction to aptamer technology and possible therapeutic applications
General scheme for a SELEX procedure
Target
DNA: Strand
separation
RNA: transcription Formation of
DNA/RNA-target
complexes
PCR or RT-PCR
amplification
Separation of
bound aptamers
Target
Target
11. Introduction to aptamer technology and possible therapeutic applications
Methods of separation
• Target binding affinity to a support (SELEX)
• Plates or resins conjugated to streptavidin, antibodies, etc.
• Colloidal gold, etc.
• Filtering using nitrocellulose membranes.
• Microfluidic systems (M-SELEX)
• Chromatography (MonoLEX)
• Centrifugation (Cell SELEX)
• Large targets (cells, viruses, etc).
• Gel electrophoresis under native conditions (PhotoSELEX)
• Capillary electrophoresis (CE-SELEX)
12. Introduction to aptamer technology and possible therapeutic applications
SELEX
Target
DNA: Strands
separation
RNA: transcription Formation of the
ssDNA/RNA-target
complexes
PCR or RT-PCR
amplification
Separation of
bound aptamers
Target
Target
13. Introduction to aptamer technology and possible therapeutic applications
Preparation of aptamers
PCR or RT-PCR
ssDNA pool dsDNA RNA pool
• Labeling with 5‘ phosphate and L-
exonuclease treatment
• Biotin labeling and separation
with streptavidin
• Thermal denaturation
• PAGE separation
• Asymmetric PCR
14. Introduction to aptamer technology and possible therapeutic applications
Polyclonal and monoclonal aptamers
3-15 rounds of selection-
amplification
Set of aptamers with
different degrees of
affinity for the target Comparable to
molecule polyclonal antibodies
Cloning and
Comparable to
characterization of
monoclonal antibodies
aptamer
15. Introduction to aptamer technology and possible therapeutic applications
Main advantages of aptamers over antibodies
Aptamers Antibodies
Aptamers are produced by chemical Antibodies often suffer from batch to batch
synthesis resulting in little or no batch to variation
batch variation
Aptamers are identified through an in vitro Requires the use of animals
process not requiring animals
Aptamers may be obtained against non- Antibodies may be obtained only against
immunogenic proteins and toxins immunogenic proteins but not against
target representing constituents of the
body and toxic substances
Denatured aptamers can be regenerated Antibodies have limited shelf life and are
within minutes, aptamers are stable to sensitive to temperature and may undergo
long term storage and can be transported denaturation
at ambient temperature
Selection conditions can be manipulated Identification of antibodies that recognize
to obtain aptamers stable in a wide range targets under conditions other than
of environmental conditions including pH physiological is not feasible
and temperature
Reporter molecules can be attached to Labelling of antibodies can cause loss in
aptamers at precise locations not involved affinity
in binding
They are not immunogenic They are usually immunogenic
Aptamers can be modified increasing their The stability of the antibodies cannot be
stability easily altered
Their small size allows for more efficient Their larger size limits their access to
entry into the cell and its compartments cellular compartments
17. Introduction to aptamer technology and possible therapeutic applications
Potential targets of the aptamers
• Aptamers are capable of binding:
– small organic or inorganic molecules
– nucleotides
– nucleic acids
– peptides and proteins
– membranes, cells and whole organisms (virus)
18. Introduction to aptamer technology and possible therapeutic applications
Applications of aptamers
• biotechnological tools
• diagnostic systems
• therapeutic agents
19. Introduction to aptamer technology and possible therapeutic applications
Applications of aptamers as therapeutic
• Aptamers targeting coagulation factors
e.g against factor IXa
• Aptamers targeting growth factors or hormones
e.g against VEGF
• Aptamers targeting antibodies involved in autoimmune diseases
e.g. auto-antibodies against nicotinic AChRs (for m gravis)
• Aptamers targeting inflammation markers
e.g against elastase
• Aptamers targeting neuropathological targets
e.g against synthetic β-amyloid peptide (Alzheimer)
• Aptamers against infectious diseases
e.g against gp120 or HA
• Aptamers targeting membrane biomarkers
e.g against CTL-4
• Aptamers targeting whole organisms
e.g against CMV
20. Introduction to aptamer technology and possible therapeutic applications
Aptamers in use or in clinical trials
21. Introduction to aptamer technology and possible therapeutic applications
Mechanism of action of MACUGEN
• Macugen is a chemically synthesized aptamer that binds to and
inhibits the function of VEGF.
• VEGF is a protein that plays an important role in the abnormal
growth of blood vessels associated with AMD (age-related
macular degeneration) or DME (diabetic macular edema).
22. Introduction to aptamer technology and possible therapeutic applications
Mechanism of action of MACUGEN
Macugen
VEGF
Endotelial cell
VEGF receptors
23. Introduction to aptamer technology and possible therapeutic applications
Projects of the laboratory
• Biotechnological or diagnostic tool
– Aptamers against proteins of Leishmania (LiKmp-11, LiH2A, LiH3, LiPABP) (Dr. Manuel Soto, Centro de Biología
Molecular Severo Ochoa-UAM, Madrid; Aptus Biotech)
– Aptamers against NL1Tc endonuclease of T. cruzi (Dr. Manuel Carlos López, Instituto de Parasitología y Biomedicina
“López Neyra”, Granada)
– Aptamers against CD3, CD4 and CD8 (Dr. Ernesto Roldán, Hospital Ramón y Cajal, Madrid)
– Aptamers against β-amiloid peptide and “tau” protein (Drs. Ginés Lifante and Juan Jiménez, Universidad Autónoma
de Madrid)
– Aptamers against bacteria (Bioapter SL)
– Aptamers against Apo A IV (Dr. Mª Dolores López Tejero. Facultad de Biología. Universidad de Barcelona y
CEREMET-UB, Barcelona; Aptus Biotech)
• Therapeutic agents
– Aptamers against proteins involved in translation (4E-BP1, eIF4E and Mnk1 kinase) (Dr. M. Elena Martín, Hospital
Ramón y Cajal, Madrid)
– Aptamers against TLR-4 (Dr. Ignacio Lizasoaín, U. Complutense, Madrid; Aptus Biotech)
– Aptamers against purinergic receptors T2X (Dr. Juan M. Gómez, Hospital Ramón y Cajal, Madrid)
– Aptamers against abscisic acid (ABA) (Dr. Elena Zocchi, Universidad de Génova, Italia)
24. Introduction to aptamer technology and possible therapeutic applications
Selection of aptamers against LiH2A
RND40 (ssDNA)
HIS-LiH2A
DNA: Strands
separation by
Ni2+ resin
thermal
denaturation
Formation of
ssDNA/RNA-target
PCR amplification 3 rounds of complexes
selection
Selection by
centrifugation
25. Introduction to aptamer technology and possible therapeutic applications
Binding affinity of SELH2A for LiH2A protein
A B
C
D SELH2A
2.5 μg/mL
10 μg/mL
5 μg/mL
500 ng BSA
500 ng
100 ng
H2A
50 ng
25 ng
-
26. Introduction to aptamer technology and possible therapeutic applications
Mapping of the SELH2A-protein interaction
A PEPTIDE
#1
SEQUENCE
MATPRSAKKAVRKSGSKSAK
C pept5
#2 SKSAKCGLIFPVGRVGGMMR
#3 GGMMRRGQYARRIGASGAVY
#4 SGAVYLAAVLEYLTAELLEL
#5 ELLELSVKAAAQSGKKRCRL pept8
#6 KRCRLNPRTVMLAARHDDDI
#7 HDDDIGTLLKNVTLSHSGVV
#8 HSGVVPNISKAMAKKKGGKK
#9 KGGKKGKATPSA
D
B 7500 b b
pept8
b
Abs 405nm
5000
c
2500 pept5
0
-
2A
pe 1
pe 2
pe 3
pe 4
pe 5
pe 6
pe 7
pe 8
9
pt
pt
pt
pt
pt
pt
pt
pt
pt
H
pe
27. Introduction to aptamer technology and possible therapeutic applications
Binding capability
A 15000
B
12500
Abs 405 nm
10000 17500
15000
12500
7500 Abs 405nm Rd3
10000 LiAPT1
LiAPT2
7500
5000 5000
2500
0
0 25 50 100 200 400 800 800
2500
H2A BSA
Protein (ng/well)
0
0 100 200 300 400 500 600 700 800 900
Protein (ng/well)
C 10000 Rd3
AptLiH2A#1
8000 AptLiH2A#2
Abs 405nm
6000
4000
2000
0
0 10 20 30 40 50 60 70
time (min)
28. Introduction to aptamer technology and possible therapeutic applications
Specificity of the SELH2A aptamers
AptLiH2A#1 AptLiH2A#2
T C N R T C N R
H2A
29. Introduction to aptamer technology and possible therapeutic applications
Sequences of aptamers from SELH2A
AptLiH2A#1 5´-GCG GAT GAA GAC TGG TGT TGT GCA ATG ATT TTT CCG GTT GAC CAG GTA GGA ATT GTA GGC CCT AAA TAC GAG CAA C-3´
AptLiH2A#2 5´-GCG GAT GAA GAC TGG TGT GGA GTC TAC CCT GTT TTC TAG TCT GCC ATC CCT ATC CCA TGC CCT AAA TAC GAG CAA C-3´
30. Introduction to aptamer technology and possible therapeutic applications
Mapping of the H2A-aptamer interaction
A PEPTIDE
#1
SEQUENCE
MATPRSAKKAVRKSGSKSAK
#2
#3
SKSAKCGLIFPVGRVGGMMR
GGMMRRGQYARRIGASGAVY
C pept5
#4 SGAVYLAAVLEYLTAELLEL
#5 ELLELSVKAAAQSGKKRCRL
#6 KRCRLNPRTVMLAARHDDDI pept8
#7 HDDDIGTLLKNVTLSHSGVV
#8 HSGVVPNISKAMAKKKGGKK
#9 KGGKKGKATPSA
B 10000
b
b
7500
b
DO 405nm
5000
b
b
2500
0
l
2A
1
2
3
4
5
6
7
8
9
ro
pt
pt
pt
pt
pt
pt
pt
pt
pt
nt
H
pe
pe
pe
pe
pe
pe
pe
pe
pe
co
Rd3 AptLiH2A#1 AptLiH2A#2
31. Introduction to aptamer technology and possible therapeutic applications
Aptamers are able to specifically bind LiH2A
A Bound B Bound
AptLiH2A#1
AptLiH2A#2
Unbound
AptLiH2A#1
AptLiH2A#2
Lysate
Unbound
Lysate
kDa M M
94
67
43
30
20 LiH2A
14 rLiH2A
29% 79% 97%
32. Introduction to aptamer technology and possible therapeutic applications
Laboratory of Aptamers (Hospital Ramón y
Cajal)
- Dr. Víctor M. González
- Dr. M. Elena Martín
- Eva M. García-Recio
- M. Isabel Pérez-Morgado
- Marta García
- Dr. Natalia Guerra (2005-2010)
- Edurne Ramos (2004-2009)
Aptus Biotech
- Dr. Gerónimo F. Gómez-Chacón
- Marta Sánchez
Centro de Biología Molecular (CSIC-UAM)
- Dr. Manuel Soto