Cell Surface Targeting

1
Cell Surface Targeting

• 8/7/06

2
Progress/agenda

• Streptavidin BioBricks
• Sequencing for QuikChange-mutagenized
  single-chain dimer streptavidin showed 141bp
  deletion 394-534
• Performed QuikChange mutagenesis on SCD
  streptavidin clones originally received in pET22
  vector. Received new streptavidin clone (pTSA13)
  from Dr. Takeshi Sano
• Plan to extract mutagenized clones and pTSA13 by
  PCR
• Lpp-OmpA assembly with streptavidin
• Ligations have been unsuccessful, possibly due to
  attempted use of NEB T4 DNA Ligase when the Roche
  Rapid DNA Ligase ran out. Also received two
  Lpp-OmpA constructs from Georgiou lab.
• Plans to retry ligations with new Rapid DNA
  ligase kit, and BioBrick-PCR Georgiou constructs
• B0032 (ribosome binding site BioBrick)
• Transformed and confirmed DNA sequence
• Plans to ligate R0010 (Lac promoter) and B0032
  upstream of Lpp-OmpA-Strep

3
Adaptamers
4
Outline

• Gel shifts
• Streptavidin beads
• New designs
• Whats next

5
Outline

• Gel shifts
• Streptavidin beads
• New designs
• Whats next

6
T20
A20
S20
T35
A35
S35
T50
Link-aptamer
A50
S50
7
Shift! (but is it due to thrombin?)
1
2
3
4
5
6
7
8
9
10
11
12

• 1 SeeBlue Plus 2 ladder
• 2 thrombin
• 3 thrombin T35
• 4 thrombin S35
• 5 thrombin T35 S35
• 6 1kb ladder
• 7 T35
• 8 T35 thrombin
• 9 S35
• 10 S35 thrombin
• 11 S35 T35
• 12 S35 T35 thrombin

188
98
62
49
38
12 polyacrylamide gel Tris-Glycine Buffer
White background stained for protein black
background stained for DNA
8
Hopefully.
1 2 3 4 5 6 7 8 9 10
11 12

• 1 SeeBlue Plus 2 ladder
• 2 thrombin
• 3 thrombin T35
• 4 thrombin S35
• 5 thrombin T35 S35
• 6 25 bp ladder
• 7 T35
• 8 T35 thrombin
• 9 S35
• 10 S35 thrombin
• 11 S35 T35
• 12 S35 T35 thrombin

188
98
62
49
38
100
75
50
25
6 polyacrylamide gel .5X TBE Buffer
Meanwhile, pure thrombin aptamer does not show a
shift. Larger weight of A35 (S35T35) may be
factor.
9
No shift for A20
1 SeeBlue Plus 2 ladder 2 thrombin 3
thrombin T50 4 thrombin S50 5 thrombin
T50 S50 6 25bp ladder 7 T50 8 T50
thrombin 9 S50 10 S50 thrombin 11 S50
T50 12 S50 T50 thrombin
188
98
62
49
38
100
75
50
25
12 polyacrylamide gel Tris-Glycine Buffer
10
Shift for A50
1 2 3 4 5 6 7 8 9
10 11 12
1 SeeBlue Plus 2 ladder 2 thrombin 3
thrombin T50 4 thrombin S50 5 thrombin
T50 S50 6 1kb ladder 7 T50 8 T50
thrombin 9 S50 10 S50 thrombin 11 S50
T50 12 S50 T50 thrombin
188
98
62
49
38
200
100
12 polyacrylamide gel Tris-Glycine Buffer
11
No shifts for streptavidin (A20, A35)
1 2 3 4 5 6 7 8 9 10
11 12
1 2 3 4 5 6 7 8 9 10
11 12
N 20 12 gel, Tris-glycine buffer
N 35 6 gel, .5X TBE

• 1 SeeBlue Plus 2 ladder
• 2 streptavdin
• 3 streptavidin TN
• 4 streptavidin SN
• 5 streptavidin TN SN
• 6 ladder

7 TN 8 TN streptavidin 9 SN 10 SN
streptavidin 11 SN TN 12 SN TN
streptavidin
12
Outline

• Gel shifts
• Streptavidin beads
• New designs
• Whats next

13
Streptavidin Beads

• First try botched control, botched washes

14
Second try
S0 streptavidin aptamer (40 bp single
stranded) SB streptavidin beads TB thrombin
beads
1 2 3 4 5
6 7 8 9

• 1 ladder
• 2 40 pmol S0
• 3 10 uL SB 40 pmol S0
• 4 25 uL SB 40 pmol S0
• 5 50 uL SB 40 pmol S0
• 6 100 uL SB 40 pmol S0
• 7 150 uL SB 40 pmol S0
• 8 T35 (50bp, single stranded oligo)
• 9 150uL TB 40 pmol S0

60
50
40
30
20
10
Above Elutions with streptavidin Below Washes
with buffer
60
50
40
30
20
10
15
Outline

• Gel shifts
• Streptavidin beads
• New designs
• Whats next

16
Designing adaptamer linkers

• Problems adaptamer linker sequences can basepair
  to aptamer regions or to self

17
Designing adaptamer linkers

• Program to evolve optimal adaptamers
• Minimize number of 4-mers that have bad
  basepairing properties
• Metropolis algorithm
• Start with random sequence, repeatedly identify
  worst 4-mer, mutate with some probability

18
Results
Thrombin aptamer
Streptavidin aptamer
T50
S50
19
New designs
Streptavidin aptamer
Thrombin aptamer
20
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21
Outline

• Gel shifts
• Streptavidin beads
• New designs
• Whats next

22
Whats next

• Ordered fluorescently labeled aptamers will try
  nitrocellulose filter assay
• Order new designs with/without fluorophores.
• Retry streptavidin-agarose bead assay with full
  adaptamers maybe even see if thrombin can bind
  together
• FRET experiment?
• Design Biacore experiment.

23
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