Cell Surface Targeting 8/7/06. Progress/agenda Streptavidin BioBricks –Sequencing for QuikChange-mutagenized single-chain dimer streptavidin showed 141bp.

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Cell Surface Targeting 8/7/06

Progress/agenda Streptavidin BioBricks –Sequencing for QuikChange-mutagenized single-chain dimer streptavidin showed 141bp deletion # –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

Adaptamers

Outline Gel shifts Streptavidin beads New designs What’s next

Outline Gel shifts Streptavidin beads New designs What’s next

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

Shift! (but is it due to thrombin?) 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 % polyacrylamide gel; Tris-Glycine Buffer White background stained for protein; black background stained for DNA

Hopefully. 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 Meanwhile, pure thrombin aptamer does not show a shift. Larger weight of A35 (=S35+T35) may be factor % polyacrylamide gel;.5X TBE Buffer

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 % polyacrylamide gel; Tris-Glycine Buffer

Shift for A50 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 % polyacrylamide gel; Tris-Glycine Buffer

No shifts for streptavidin (A20, A35) 1: SeeBlue Plus 2 ladder 2: streptavdin 3: streptavidin + TN 4: streptavidin + SN 5: streptavidin + TN + SN 6: ladder N= 20; 12% gel, Tris-glycine buffer 7: TN 8: TN + streptavidin 9: SN 10: SN + streptavidin 11: SN + TN 12: SN + TN + streptavidin N= 35; 6% gel,.5X TBE

Outline Gel shifts Streptavidin beads New designs What’s next

Streptavidin Beads First try: botched control, botched washes

Second try 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 S0: streptavidin aptamer (40 bp single stranded) SB: streptavidin beads TB: thrombin beads Above: Elutions with streptavidin Below: Washes with buffer

Outline Gel shifts Streptavidin beads New designs What’s next

Designing adaptamer linkers Problems: adaptamer linker sequences can basepair to aptamer regions or to self

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

Results S50 T50 Streptavidin aptamer Thrombin aptamer

New designs Thrombin aptamer Streptavidin aptamer

Outline Gel shifts Streptavidin beads New designs What’s 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.