Aptamers in the Real World Development and Applications

1. Aptamers in the Real WorldDevelopment and Applications NeoVentures Biotechnology Inc. www.neoventures.ca Aptamers Applied

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. Aptamers Applied

3. Reasons why aptamers do not work well in diagnostics. • They are not selected for appropriate specificity. • They are not selected for immobilization. • They fold down onto surfaces and become inactive. • They do not bind with sufficient affinity. Aptamers Applied

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

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

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

7. High throughput binding assays Immobilize multiple candidate sequences onone chip. Flow target molecule over sequences. Assess binding kinetics on all sequences simultaneously. Horiba OpenplexSPRi Aptamers Applied

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

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

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

11. Demonstration with thrombin aptamer. Immobilized aptamerkD = 207 nM Anchor immobilized kD = 38 nM Aptamers Applied

12. 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] Aptamers Applied

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

14. Validation with aflatoxinaptamer Aflatoxinaptamer Measurement of captured aflatoxin B1 fluorescence. Aptamers 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. Aptamers Applied

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

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

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

22. Aptamer advantages for diagnostic producers. Aptamers Applied

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

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