1. Aptamers in the Real World Development and Applications NeoVentures Biotechnology Inc. www.neoventures.ca Aptamers Applied1

2. 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. 2Aptamers Applied

3. 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. 3Aptamers Applied

4. Appropriate specificity Select for the desired target form. Select against other forms of the target. 4Aptamers Applied

5. 5 End point sequencing Dynamic-Deep Sequencing Selection Rounds Deep sequence every round Traditional Dynamic-Deep 200 sequences 1 million sequences per selection round

6. Aptamers Applied6 Non-linear selection Standard selection Positive selection against negative target Positive selection Positive selection against matrix vs Candidate aptamers Sequence analysis with contrasts:

7. 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 7Aptamers Applied

8. Immobilized binding Aptamers must bind while immobilized to function. – We do not select for this, we screen for it. 8Aptamers Applied

9. Hydrostatic interactions Aptamers fold down onto charged surfaces. 9Aptamers Applied

10. Solution Extension of aptamer Anchor Double stranded base constrains fold down while maintaining binding capacity. 10Aptamers Applied

11. Demonstration with thrombin aptamer. Immobilized aptamerk D = 207 nM Anchor immobilized k D = 38 nM 11Aptamers Applied

12. Sensitivity limited by binding affinity of aptamers. Yes and no... This means that affinity (k a ) is equivalent to aptamer concentration [A]. – 10X lower binding affinity can be compensated for by 10X higher concentration of ligand. d[AT]/dt = k a [A][T] – k d [AT] 12Aptamers Applied

13. Increasing the concentration of capture aptamer BSA 13Aptamers Applied Anneal aptamers to anchors. Conjugate anchors to protein (BSA). Passively immobilize BSA on surface.

14. Validation with aflatoxin aptamer Aflatoxin aptamer Measurement of captured aflatoxin B1 fluorescence. 14Aptamers Applied

15. Washing is also a problem Wash = new equilibrium Weak binding = target loss Target loss = poor sensitivity This is more important than initial capture. 15Aptamers Applied

16. Docking is an issue Large targets will block available aptamers when docked. Increasing concentration works best with small molecules 16Aptamers Applied

17. Combined capture aptamers Two aptamers for different epitopes on target protein. Mixed together on capture surface. Combined binding increases affinity. 17Aptamers Applied

18. Multiple aptamers for detection Multiple detection aptamers -All labeled with the same fluorophore. -Bind to different epitopes on the same captured protein. (simultaneously). 18Aptamers Applied

19. 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. 19Aptamers Applied

20. Commercial considerations The market is not looking for innovation. The market wants more fish and they want the fishing to be easier. 20Aptamers Applied

21. 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 21Aptamers Applied

22. Aptamer advantages for diagnostic producers. 22Aptamers Applied

23. The future for antibodies Aptamers Applied23 Improvements in cost and convenience are disruptive.

24. Thank you Aptamers Applied www.neoventures.ca 24Aptamers Applied