1. Cell Surface Targeting: The week of waiting 8/14/06

2. Adaptamers A20 A35 A50 Link-aptamer T35 S35 S50 T20 S20 T50

3. Outline Thrombin aptamer/thrombin beads A20 experiment Magnetic beads

4. Thrombin aptamer 1: dsDNA ladder 2: 20 pmol T0 3: 10 uL TB + 80 pmol T0 4: 25 uL TB + 80 pmol T0 5: 50 uL TB + 80 pmol T0 6: 100 uL TB + 80 pmol T0 7: 150 uL TB + 80 pmol T0 8: 150 uL TB + S5 (50bp, single stranded oligo) 9: 150uL SB + 80 pmol T0 1 2 3 4 5 6 7 8 9 T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TB: thrombin beads 10 30 20 40 50 10 20 40 50 30 Above: Elutions with thrombin Below: Washes with buffer

5. A20 experiment Incubated adaptamer A20 and thrombin with streptavidin beads; washes and elutions using free streptavidin were collected. As controls, streptavidin beads + thrombin were also incubated with T20 or S20, and trypsin beads + thrombin were incubated with A20. A20 T20 S20

6. A20 Experiment: DNA 1: ladder 2: 20 pmol T20 3: 20 pmol S20 4: 20 pmol A20 5-8: SB + A20 + varying concentrations of thrombin 9: SB + T20 + 320 pmol thrombin 10: SB + S20 + 320 pmol thrombin 11: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 1 2 3 4 5 6 7 8 9 10 11 T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads Above: Elutions with streptavidin Below: Washes with buffer 10 50 30 40 20 10 50 40 20 30

7. A20 Experiment: Protein 1: ladder 3: 40 pmol thrombin 4:SB + A20 + 40 pmol thrombin 5:SB + A20 + 80 pmol thrombin 6:SB + A20 + 160 pmol thrombin 7:SB + A20 + 320 pmol thrombin 8: SB + T20 + 320 pmol thrombin 9: SB + S20 + 320 pmol thrombin 10: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 1 2 3 4 5 6 7 8 9 10 Above: Elutions with streptavidin Below: Washes with buffer “250” “148” “98” “64” “250” “148” “98” T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads

8. Elutions Pt. 2: Silver Staining 1: ladder 3: 40 pmol thrombin 4:SB + A20 + 40 pmol thrombin 5:SB + A20 + 80 pmol thrombin 6:SB + A20 + 160 pmol thrombin 7:SB + A20 + 320 pmol thrombin 8: SB + T20 + 320 pmol thrombin 9: SB + S20 + 320 pmol thrombin 10: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 1 2 3 4 5 6 7 8 9 10 T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads

9. Elutions Pt. 3: SDS-PAGE 1 2 3 4 5 6 7 8 9 10 1: ladder 2: 20 pmol thrombin 3: 20 pmol streptavidin 4:SB + A20 + 40 pmol thrombin 5:SB + A20 + 80 pmol thrombin 6:SB + A20 + 160 pmol thrombin 7:SB + A20 + 320 pmol thrombin 8: SB + T20 + 320 pmol thrombin 9: SB + S20 + 320 pmol thrombin 10: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 250 148 98 64 50 36 22 16 6 4 T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads

10. L T S 1 2 3 4 5 6 7 250 148 98 64 50 36 22 16 6 4 “250” “148” “98” “64” “250” “148” “98” “64” L: ladder 1:SB + A20 + 40 pmol thrombinT: thrombin 2:SB + A20 + 80 pmol thrombinS: streptavidin 3:SB + A20 + 160 pmol thrombin 4:SB + A20 + 320 pmol thrombin 5: SB + T20 + 320 pmol thrombin 6: SB + S20 + 320 pmol thrombin 7: TrB + A20 + 320 pmol thrombin L T 1 2 3 4 5 6 7 12% Native PAGE12% SDS-PAGE T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads

11. By the way.. 1: Seeblue plus2 ladder 2: BSA 3: human thrombin (same used in experiment) 4: Sigma human thrombin 5: Sigma bovine thrombin 6: streptavidin 250 148 98 64 50 36 16 6 4

12. Magnetic Beads Received streptavidin magnetic beads Incubated magnetic beads with A50 or S0, then added thrombin beads. Magnetic and thrombin beads clump together!

14. Streptavidin beads +Control: streptavidin beads+ thrombin beads +A50 thrombin beads + S0

15. Control: beads + S0 Beads + A50

18. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

19. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

20. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

21. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

22. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

23. Streptavidin beads + A50 Control: Streptavidin beads + S0

24. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

25. Streptavidin beads +Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

26. Next: Get new adaptamers! Wash streptavidin beads; run on gel. Retry bead assay with new adaptamers. Try mixing streptavidin agarose and magnetic beads with biotinylated oligos. At the very least, run gel which shows that adaptamers can bind thrombin and streptavidin beads separately.