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Aptamers in the Real World

Development and ApplicationsNeoVentures Biotechnology

Inc.www.neoventures.ca

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NeoVentures was the first to commercialize aptamer based

diagnostics

Aptamer based affinity columns formycotoxins.

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 desiredtarget form.

Select against other formsof the target.

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End pointsequencing

Dynamic-Deep Sequencing

Selection Rounds

Deep sequence every round

Traditional

Dynamic-Deep

200 sequences

1 million sequences per selection round

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Non-linear selection

Standard selection

Positive selection against negative target

Positive selection

Positive selection against matrix

vs

vsCandidate aptamers

Sequence analysis with contrasts:

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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

Extension of aptamerAnchor

Double stranded base constrains fold down while maintaining binding capacity.

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Demonstration with thrombin aptamer.

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SPRi

uni

ts

Time (seconds)

Anchored Thr

Thr

Immobilized aptamer kD = 207 nM Anchor immobilized kD = 38 nM

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Sensitivity limited by binding affinity of aptamers.

• Yes and no...

• 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.

d[AT]/dt = ka [A][T] – kd [AT]

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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

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1200

1400

0 20 40 60 80 100 120 140

Rela

tive

fluor

esce

nce

units

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 thaninitial capture.

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Docking is an issueLarge targets willblock availableaptamers whendocked.

Increasingconcentration works best with small molecules

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Combined capture aptamers

Two aptamers for different epitopes on target protein.

Mixed together on capturesurface.

Combined binding increasesaffinity.

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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 considerationsThe market is not looking for innovation.

The market wants morefish 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 AptamersProduction time 6 months 1 weekQuality Assurance Extensive SimpleInventory Absolutely Just-in-timeShipping -4 C Ambient

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The future for antibodies

Improvements in cost and convenience are disruptive.

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Thank you

Aptamers Appliedwww.neoventures.ca