1. Cell Surface Targeting
7/24/06

2. Adaptamers

3. Questions Can we observe a gel shift with the conditions we’re using?
If so, are our aptamers binding protein?

4. Answers Yes, we can observe streptavidin binding biotin.
No, neither of our aptamers appear to bind their targets. But we’re pretty sure of the reason.

5. (Very) High Concentrations
T5: thrombin aptamer + 5 nts
S5: streptavidin aptamer + 5 nts
Protein staining
DNA staining
Protein staining
2: .1% BSA
3: thrombin in .1% BSA
4: thrombin + T5 in .1% BSA
5: streptavidin + S5
6: streptavidin + S5
7: streptavidin + biotinylated oligos
DNA staining
9: biotinylated oligos
10: biotinylated oligos + streptavidin
11: S5
12: S5 + streptavidin

6. Findings 1) Observation of streptavidin binding biotin.
Protein staining
DNA staining
Protein staining
2: .1% BSA
3: thrombin in .1% BSA
4: thrombin + T5 in .1% BSA
5: streptavidin + S5
6: streptavidin + S5
7: streptavidin + biotinylated oligos
DNA staining
9: biotinylated oligos
10: biotinylated oligos + streptavidin
11: S5
12: S5 + streptavidin

7. Findings Observation of streptavidin binding biotin.
Streptavidin is not binding S5.
Protein staining
DNA staining
Protein staining
2: .1% BSA
3: thrombin in .1% BSA
4: thrombin + T5 in .1% BSA
5: streptavidin + S5
6: streptavidin + S5
7: streptavidin + biotinylated oligos
DNA staining
9: biotinylated oligos
10: biotinylated oligos + streptavidin
11: S5
12: S5 + streptavidin

8. Findings Observation of streptavidin binding biotin.
Streptavidin is not binding S5.
BSA is actually responsible for bands in lanes with thrombin.
Protein staining
2: .1% BSA
3: thrombin in .1% BSA
4: thrombin + T5 in .1% BSA
5: streptavidin + S5
6: streptavidin + S5
7: streptavidin + biotinylated oligos
DNA staining
9: biotinylated oligos
10: biotinylated oligos + streptavidin
11: S5
12: S5 + streptavidin

9. Findings Observation of streptavidin binding biotin.
Streptavidin is not binding S5.
BSA is actually responsible for bands in lanes with thrombin.
Is there a thrombin shift? Unclear.
Protein staining
2: .1% BSA
3: thrombin in .1% BSA
4: thrombin + T5 in .1% BSA
5: streptavidin + S5
6: streptavidin + S5
7: streptavidin + biotinylated oligos
DNA staining
9: biotinylated oligos
10: biotinylated oligos + streptavidin
11: S5
12: S5 + streptavidin

10. Moderate Concentration
4) Thrombin shift? No.
Protein staining
DNA staining
Protein staining
2: thrombin
3: thrombin + T5
4: streptavidin + S5
5: streptavidin + S5
6: streptavidin + biotinylated oligos
DNA staining
8: nothing + loading dye
9: T5
10: T5 + thrombin
11: S5
12: S5 + streptavidin

11. Moderate Concentration
Question: What is responsible for these bands?
Protein staining
DNA staining
Protein staining
2: thrombin
3: thrombin + T5
4: streptavidin + S5
5: streptavidin + S5
6: streptavidin + biotinylated oligos
DNA staining
8: nothing + loading dye
9: T5
10: T5 + thrombin
11: S5
12: S5 + streptavidin

12. More answers Have been using bovine thrombin, not human thrombin.
Secondary structure issues: everyone else denatures their aptamers prior to incubation with protein

13. Next: Change the thrombin, add denaturation If it works,
try adaptamer experiments; also, redesign adaptamers to avoid secondary structure conflicts.
Order aptamers that can bind a cell.
If it doesn’t,
Put on thinking cap.

15. Sequencing results Clones from Ting lab: StrepW, StrepH, StrepD
BioBrick’d and sent out for sequencing last week
Results
StrepW: Correct sequence from both forward/reverse reactions
StrepH: One mutation at bp 344, T to C
GCT to GCC, silent mutation for alanine
StrepD: Correct sequence from forward reaction, correct sequence from reverse reaction
Performed midipreps

16. BioBricks for Lpp-OmpA
OmpA PCR
46-66
100
1000
400
200
500
1650
46-159
full
300
Lpp PCR
100
400
200
500
300
1-29
full
+stop
XbaI/PstI digest
Lpp
1-29
OmpA
46-66
46-159
100
400
200
500
300

17. BioBricks for SCD streptavidin
Single-chain dimer clones from Aslan lab
SCD-NM C2 E2
E X
StrepSCDF
StrepSCDMF
StrepSCDMR 1
825
S P
StrepSCDR
PstI site, 620
CTGCAG CTGCGG
100
400
200
500
300
SCD-NM C E2
MF/R
F/MR
F/R
non-mut
650
850
1000
1st PCR
400
300
SCD-NM C E2
F/R
Crossover PCR
sent out for
sequencing

18. Sequencing results Homology in bp regions ~1-250 and ~550-800 Why?
Single
Chain
Dimer
Forward primer annealing region
Bp : gaggccaacgccaagaagtc
Bp : gaggccaacgcctggaagtc
Explains double PCR products with F/MR and F/R primers
Does not explain single crossover PCR product

19. Progress/plans Midipreps of StrepW, StrepH, StrepD
BioBricks of Lpp(1-29), OmpA(46-66) and (46-159); sent out for sequencing
Confirm sequences of Lpp and OmpA parts; midiprep.
Digest and assembly.
Figure out solution for StrepSCD PCR
Design new primers
Anneal upstream on plasmid
PCR in separate parts and assemble