Cell Surface TargetingCell Surface Targeting

7/24/06

AdaptamersAdaptamers

QuestionsQuestions

Can we observe a gel shift with the conditions we’re using?

If so, are our aptamers binding protein?

AnswersAnswers

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.

(Very) High Concentrations(Very) High Concentrations

S5: streptavidin aptamer + 5 nts

T5: thrombin aptamer + 5 nts

Protein staining2: .1% BSA 3: thrombin in .1% BSA4: thrombin + T5 in .1% BSA5: streptavidin + S56: streptavidin + S57: streptavidin + biotinylated oligosDNA staining9: biotinylated oligos 10: biotinylated oligos + streptavidin11: S512: S5 + streptavidin

Protein staining DNA staining

FindingsFindings

Protein staining2: .1% BSA 3: thrombin in .1% BSA4: thrombin + T5 in .1% BSA5: streptavidin + S56: streptavidin + S57: streptavidin + biotinylated oligosDNA staining9: biotinylated oligos 10: biotinylated oligos + streptavidin11: S512: S5 + streptavidin

1) Observation of streptavidin binding biotin.

Protein staining DNA staining

FindingsFindings

Protein staining2: .1% BSA 3: thrombin in .1% BSA4: thrombin + T5 in .1% BSA5: streptavidin + S56: streptavidin + S57: streptavidin + biotinylated oligosDNA staining9: biotinylated oligos 10: biotinylated oligos + streptavidin11: S512: S5 + streptavidin

1) Observation of streptavidin binding biotin.2) Streptavidin is not binding S5.

Protein staining DNA staining

FindingsFindings

Protein staining2: .1% BSA 3: thrombin in .1% BSA4: thrombin + T5 in .1% BSA5: streptavidin + S56: streptavidin + S57: streptavidin + biotinylated oligosDNA staining9: biotinylated oligos 10: biotinylated oligos + streptavidin11: S512: S5 + streptavidin

1) Observation of streptavidin binding biotin.2) Streptavidin is not binding S5.3) BSA is actually responsible for bands in lanes with thrombin.

FindingsFindings

Protein staining2: .1% BSA 3: thrombin in .1% BSA4: thrombin + T5 in .1% BSA5: streptavidin + S56: streptavidin + S57: streptavidin + biotinylated oligosDNA staining9: biotinylated oligos 10: biotinylated oligos + streptavidin11: S512: S5 + streptavidin

1) Observation of streptavidin binding biotin.2) Streptavidin is not binding S5.3) BSA is actually responsible for bands in lanes with thrombin.4) Is there a thrombin shift? Unclear.

Moderate ConcentrationModerate Concentration

Protein staining2: thrombin 3: thrombin + T54: streptavidin + S55: streptavidin + S56: streptavidin + biotinylated oligosDNA staining8: nothing + loading dye 9: T510: T5 + thrombin11: S512: S5 + streptavidin

4) Thrombin shift? No.

Protein staining DNA staining

Moderate ConcentrationModerate Concentration

Protein staining2: thrombin 3: thrombin + T54: streptavidin + S55: streptavidin + S56: streptavidin + biotinylated oligosDNA staining8: nothing + loading dye 9: T510: T5 + thrombin11: S512: S5 + streptavidin

Question: What is responsible for these bands?

Protein staining DNA staining

More answersMore answers

Have been using bovine thrombin, not human thrombin.

Secondary structure issues: everyone else denatures their aptamers prior to incubation with protein

Next:Next:

Change the thrombin, add denaturationIf 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.

Sequencing resultsSequencing results Clones from Ting lab: StrepW, StrepH, StrepD BioBrick’d and sent out for sequencing last week

Results– StrepW: Correct sequence 1-444 from both forward/reverse reactions– StrepH: One mutation at bp 344, T to C

GCT to GCC, silent mutation for alanine– StrepD: Correct sequence 1-409 from forward reaction, correct sequence

410-444 from reverse reaction

Performed midipreps

BioBricks for Lpp-OmpABioBricks 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 full+stop

XbaI/PstI digest

Lpp1-29

OmpA46-66

OmpA46-159

100

400

200

500

300

BioBricks for SCD streptavidinBioBricks for SCD streptavidin Single-chain dimer

clones from Aslan lab– SCD-NM– C2– E2

E XStrepSCDF

S PStrepSCDR

StrepSCDMF

StrepSCDMR

1 825

PstI site, 620CTGCAG CTGCGG

100

400

200

500

300

SCD-NM C2 E2

MF/R

F/MR

F/Rnon-mut

650850

1000

1st PCR

400

300

SCD-NM C2 E2

F/R

Crossover PCR

sent out for sequencing

Sequencing resultsSequencing results Homology in bp regions ~1-250 and ~550-800 Why?

– Single– Chain– Dimer

Forward primer annealing region– Bp 004-023: gaggccaacgccaagaagtc– Bp 538-557: gaggccaacgcctggaagtc

Explains double PCR products with F/MR and F/R primers Does not explain single crossover PCR product

Progress/plansProgress/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