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
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