as New T ools for I nhibiting Cytokine Activity Howard A Young Laboratory of Experimental Immunology Cancer and Inflammation Program National Cancer Institute at Frederick Frederick MD ID: 433395
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Slide1
Aptamers as New Tools for Inhibiting Cytokine Activity
Howard A. YoungLaboratory of Experimental ImmunologyCancer and Inflammation ProgramNational Cancer Institute at FrederickFrederick, MD Slide2
Collaboration with:
Nucleic Acid Synthetic Biology
Resarch
Team
Systems and structural Biology Center
Yokohama Institute, RIKEN
Ichiro Hirao
Team Leader
RIKEN Center for Life Science Technologies
President & CEO
TagCyx
BiotechnologiesSlide3
Aptamers (from the Latin aptus - fit, and Greek
meros - part) are oligonucleic acid or peptide molecules that bind to a specific target molecule. Slide4
Nat Biotechnol. 2013 May;31(5):453-7. doi: 10.1038/nbt.2556. Epub
2013 Apr 7.Generation of high-affinity DNA aptamers using an expanded genetic alphabet.Kimoto M1, Yamashige R,
Matsunaga K
,
Yokoyama S
,
Hirao
I
.AbstractDNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins.
Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo
[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.Slide5
Genetic alphabet expansion of the central dogma by unnatural base pairs
DNA aptamers, PCR detection
RNA labeling, functional RNAs
Unnatural proteins
Next-generation
biotechnology by
synthetic
xenobiology
In vivo systems
Ds
Px
Diagnostic
and
therapeutic
applications
Nat. Methods
, 3, 729 (2006).
Nucleic Acids Res
., 37, e14 (2009
).
Nucleic Acids Res
., 40, 2793 (2012
).Slide6
DNA aptamer selection for protein targets
Selection
PCR amplification
DNA aptamer
Conventional DNA library
Conventional aptamer selection
Selection
PCR amplification
DNA aptamer
New DNA library
with five different bases
Unnatural base
New methods using unnatural base pair systems
H
igh affinity ?
In vitro
selection (SELEX)
A.D. Ellington & J.W.
Szostak
, Nature, 346, 818 (1990
).
C
.
Tuerk
& L. Gold, Science, 249, 505 (1990
).
D.L
. Robertson & G.F. Joyce, Nature, 344, 467 (1990).Slide7
Systematic Evolution of
Ligands by Exponential EnrichmentSELEX involving the Ds−Px
pair
Ds
Px
Selection
PCR amplification
DNA aptamer
Target protein
Nature
Biotechnol
.,
31
, 453-457 (2013).
2
）
Increase in structural diversity
because of no pairing partner of Ds
１）
Increase in hydrophobicity
DNA
aptamer
Hydrophobic Ds
Target protein
Hydrophobic pocketSlide8
High affinity aptamer
generation by
an unnatural base pair system
Selection
PCR
amplification
Target
protein
Ds-aptamer : protein complex
Ds-containing DNA library
SELEX
Ds-base
New nucleic acid antibodies
The binding abilities represented more than a 100-fold increase compared with thos
e for conventional DNA aptamers.
Nature
Biotechnol
.,
31
, 453-457 (2013).Slide9
Anti-IFNγ DNA aptamer selection
Ds
T
T
C
C
12
T
A
G
G
T
T
G
G
G
G
T
G
T
A
G
G
G
C
C
G
G
A
T
C
T
A
Ds
T
T
17
18
T
T
A
A
29
A
Ds
40
G
A
c
t
c
a
c
49
1
8
26
33
Stem-1
Stem-2
G
G
41
38
37
32
31
27
9
10
>96%
>99%
<80%
: DMS
: DMS, protected by protein binding
: KMnO
4
: KMnO
4
, protected by protein binding
: KMnO
4
,
enhanced by protein binding
:
MungBean
nuclease,
p
rotected by
protein binding
Base conservation
after the doped selection
Chemical and enzymatic probing
:
MungBean
nuclease
Anti-IFN
γ
Ds x 3 (49-mer)
Nature
Biotechnol
.,
31
, 453-457 (2013).Slide10
Anti-IFNγ DNA aptamer selection
Tm = 37.6 ˚C
Tm = 33.1 ˚C
Ds
Ds
Ds
Anti-IFN
γ
Ds x 3
(49-mer)
K
D
=
0.038
nM
38
pM
Nature
Biotechnol
.,
31
, 453-457 (2013).
20
30
10
0
Response units (RU)
0
200
400
Time (sec)
2.5
50
30
20
10
5
IFNγ
(nM)
1.25
A
A
A
Anti-IFN
γ
A x 3
(49-mer)
K
D
=
7.21
nM
0
200
400
600
Time (sec)
600
Ramanathan
, M. et al.,
Transplantation
,
52
, 612 (1994).
K
nown anti-IFN
γ
DNA aptamer
(26-mer)
K
D
=
16.6
nM
100
150
5
0
0
Time (sec)
0
300
600
900
30
nM
2
0
nM
1
0
nM
5
nM
2.5
nM
IFN-γSlide11
Anti-IFNγ DNA aptamer selection
Protein 20
nM
100
150
50
0
200
250
0
300
600
900
Normalized Responses
High selectivity of the anti-IFN
γ
aptamer (aptamer Ds x 3, 49-mer)
VEGF
165
BSA
VEGF
121
IFN
γ
EGF
Thrombin
Time (sec)
Sensorgrams
(SPR) for the binding of different proteins (20
nM
)
to 5′-biotinylated aptamer Ds x 3 (49-mer)
in 1×PBS + 0.05% NP40Nature
Biotechnol., 31, 453-457 (2013).Slide12
Assay for Biological ActivityTreat target cell (human breast tumor cell line) with IFN-
g for 10 minutesAssay for phospho-STAT1 via flow cytometry Slide13
Aptamer effects on IFNγ induced pSTAT1 at 37˚C for 10 min
2 ng
10 ng
100
ng
38
pM
IFN
γ
= 2 ng
Aptamer
addition
Unpublished data
2 ng
10 ng
100
ng
16
nM
Anal. Chem
.,
82
, 1851 (2010).Slide14
Nuclease degradation and stabilization of aptamers
10 min
37˚C
O/N
r.t.
O/N
37˚C
38
pM
10 min
37˚C
O/N
r.t.
O/N
37˚C
5’
3’
Unpublished dataSlide15
Conclusions:
1. IFN-
g
aptamer
interaction is extremely rapid
2.
Aptamer
blocks IFN-
g
interaction with cell surface receptor
DNA-
Aptamer
Time Course and Effect of Wash out and
RestimulationSlide16
Stabilization of DNA aptamers by attaching a mini-hairpin DNA sequence
Ds
Ds
Ds
Ds
Ds
Ds
0
1
6
24
0
1
6
24
48
72
48
72
(
hr
)
In 96
％
human serum
Ds-DNA aptamer
Ds-DNA aptamer
+
Mini-hairpin DNA
Mini-hairpin DNA
Unpublished dataSlide17
Stability of the mini-hairpin DNAs against nucleasesSlide18
Extraordinarily thermal stable mini-hairpin DNA (
dGCGAAGC)
Nucleic Acids Res.,
17
, 2223
(
1989) and
22
, 576 (1994).
Tm = 76.5 ˚CSlide19
Aptamer (100 ng/ml) effects on IFNγ induced pSTAT1 at 37˚C, overnight
Kd
= 16
nM
Unpublished data
200
ng/mlSlide20
Diagnostic and therapeutic applications of new DNA aptamers20
Ds
Ds
Ds
Unnatural-base DNA aptamers
Mini-hairpin DNA
High stable and high affinity
DNA aptamers
for imaging, diagnostics and therapeuticsSlide21
AcknowledgementsRIKEN Center for Life Science Technologies
TagCyx BiotechnologiesDr. Ichiro Hirao
NCI at Frederick
Charlotte Hanson
Michael
S
anford