Aptamers is the real world: problems and opportunities

Report
Aptamers in the Real World
Development and Applications
NeoVentures Biotechnology Inc.
www.neoventures.ca
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NeoVentures was the first to
commercialize aptamer based diagnostics
Aptamer based affinity columns for
mycotoxins.
Aptamer (fluorescence polarization)
kits for protein quality in wheat.
We focus now on providing aptamer identification
and application services for others.
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Reasons why aptamers do not work
well in diagnostics.
1. They are not selected for appropriate
specificity.
2. They are not selected for immobilization.
3. They fold down onto surfaces and become
inactive.
4. They do not bind with sufficient affinity.
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Appropriate specificity
Select for the desired
target form.
Select against other forms
of the target.
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Dynamic-Deep Sequencing
Traditional
Selection Rounds
End point
sequencing
200 sequences
Dynamic-Deep
Deep sequence every round
1 million sequences per selection round
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Non-linear selection
Positive selection against negative target
Standard selection
Positive selection
Positive selection against matrix
Sequence analysis with contrasts:
vs
Candidate aptamers
vs
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High throughput binding assays
Immobilize multiple candidate sequences
on one chip.
Flow target molecule over sequences.
Assess binding kinetics on all sequences
simultaneously.
Horiba Openplex SPRi
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Immobilized binding
• Aptamers must bind while immobilized to
function.
– We do not select for this, we screen for it.
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Hydrostatic interactions
Aptamers fold down onto
charged surfaces.
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Solution
Anchor
Extension of aptamer
Double stranded base constrains fold down
while maintaining binding capacity.
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Demonstration with thrombin
aptamer.
12
10
SPRi units
8
6
Anchored Thr
4
Thr
2
0
0
100
-2
Immobilized aptamer
Anchor immobilized
200
300
400
500
Time (seconds)
kD = 207 nM
kD = 38 nM
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Sensitivity limited by binding affinity of
aptamers.
• Yes and no...
d[AT]/dt = ka [A][T] – kd [AT]
• This means that affinity (ka ) is equivalent to
aptamer concentration [A].
– 10X lower binding affinity can be compensated for
by 10X higher concentration of ligand.
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Increasing the concentration of
capture aptamer
BSA
• Anneal aptamers to anchors.
• Conjugate anchors to protein (BSA).
• Passively immobilize BSA on surface.
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Validation with aflatoxin aptamer
Relative fluorescence units
1400
1200
1000
800
600
400
200
0
0
20
40
60
80
100
120
140
Aflatoxin concentration (nM)
Aflatoxin aptamer
Measurement of captured aflatoxin B1 fluorescence.
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Washing is also a problem
Wash = new equilibrium
Weak binding = target loss
Target loss = poor sensitivity
This is more important than
initial capture.
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Docking is an issue
Large targets will
block available
aptamers when
docked.
Increasing
concentration works
best with small
molecules
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Combined capture aptamers
Two aptamers for
different epitopes on
target protein.
Mixed together on capture
surface.
Combined binding increases
affinity.
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Multiple aptamers for detection
Multiple detection aptamers
-All labeled with the same
fluorophore.
-Bind to different epitopes on the
same captured protein.
(simultaneously).
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Small size of aptamers
• Used to bind to multiple epitopes
simultaneously without physical constraint.
– Advantage of aptamers over antibodies
– Increase binding affinity
– Increase signal
• Next generation sequencing enables
identification of the multiple aptamers
required.
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Commercial considerations
The market is not looking
for innovation.
The market wants more
fish and they want the
fishing to be easier.
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It is given
• Antibodies do not cost very much.
– Aptamers must cost less, but this is not an advantage.
• Diagnostic tests cannot be more complicated to
perform.
– Learning something new is a barrier.
• No need for new capital equipment
– Kits must use existing reading equipment.
• Fluorescence
• Colour
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Aptamer advantages for diagnostic
producers.
Antibodies
Production time
Quality Assurance
Inventory
Shipping
6 months
Extensive
Absolutely
-4 C
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Aptamers
1 week
Simple
Just-in-time
Ambient
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The future for antibodies
Improvements in cost and
convenience are disruptive.
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Thank you
Aptamers Applied
www.neoventures.ca
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