190
1. Prepare a 0.75-mm-thick 12% resolving gel by mixing 4 mL
30% (w/v) acrylamide, 2.5 mL 1.5 M Tris–HCl pH 8.9,
3.3 mL dH 2 O, 100 μL 10% (w/v) SDS, 100 μL 10% (w/v)
ammonium persulfate and 6 μL TEMED. Cast the gel up to
2 cm below the top and cover with a layer of isopropanol.
Allow the gel to polymerize for 30 min.
2. Prepare a stacking gel by mixing 830 μL 30% (w/v) acrylamide, 630 μL 1 M Tris–HCl pH 6.8, 3.4 mL dH 2 O, 50 μL
10% (w/v) SDS, 50 μL 10% (w/v) ammonium persulfate, and
3 μL TEMED. Pour the mixture on the top of the resolving
gel and insert the comb. Allow the gel to polymerize for
30 min.
3. To ensure efficient complex formation and inhibition of the
enzyme in the assembled complex, sequentially mix ODN1TEM1- β-lactamase, ssDNA template (with a target recognition sequence length of 0, 10, 20, 30, 40, or 50 nucleotides)
and ODN2-BLIP in a 1:1.2:2 ratio at low micromolar concentrations in complexation buffer and incubate for 30 minutes at
room temperature.
4. Mix the hybridized samples prepared in step 2 with an equal
volume of 2× sample buffer and load it on a semi-native 12%
SDS-PAGE gel prepared in step 1. Run the gel for 15 min at
90 V followed by 1 h at 125 V.
5. Remove the gel from the glass plates and wash with dH 2 O for
5 min. Subsequently stain the gel with Coomassie blue for
30 min. Then destain the gel with dH 2 O until clear bands
appear and image the gel (Fig. 4).
1. Mix ODN1-TEM1-β-lactamase, ODN2-BLIP, and ssDNA
template (with target recognition sequences of 0, 10, 20, 30,
40, and 50 nucleotides) in PBS
+
at low micromolar concentrations in a ratio of 1:2:1.2 and incubate for 30 min at room
temperature.
2. Dilute the mixtures in PBS
+
to a final concentration of 50 nM
ODN1-TEM1-β-lactamase.
3. In a 96-well plate, mix 4 μL of the diluted complexes with
152 μL PBS
+
and 24 μL target oligonucleotides (from 100 nM
stock solution in PBS
+
) and incubate for 1 h at room
temperature.
4. Add 20 μL nitrocefin (from 500 μM stock solution in PBS)
yielding a final concentration of 1 nM DNA-directed enzymeinhibitor complex, 12 nM target oligonucleotide, and 50 μM
nitrocefin.
5. Directly place the plate in the platereader and proceed with
measuring the optical absorbance in time of the formed product
at 486 nm.
3.4 Semi-native
SDS-PAGE
of Reconstituted
DNA-Directed EnzymeInhibitor Complex
3.5 Enzyme Activity
Assay of Reconstituted
DNA-Directed EnzymeInhibitor Complex
Wouter Engelen and Maarten Merkx
1. Prepare a 0.75-mm-thick 12% resolving gel by mixing 4 mL
30% (w/v) acrylamide, 2.5 mL 1.5 M Tris–HCl pH 8.9,
3.3 mL dH 2 O, 100 μL 10% (w/v) SDS, 100 μL 10% (w/v)
ammonium persulfate and 6 μL TEMED. Cast the gel up to
2 cm below the top and cover with a layer of isopropanol.
Allow the gel to polymerize for 30 min.
2. Prepare a stacking gel by mixing 830 μL 30% (w/v) acrylamide, 630 μL 1 M Tris–HCl pH 6.8, 3.4 mL dH 2 O, 50 μL
10% (w/v) SDS, 50 μL 10% (w/v) ammonium persulfate, and
3 μL TEMED. Pour the mixture on the top of the resolving
gel and insert the comb. Allow the gel to polymerize for
30 min.
3. To ensure efficient complex formation and inhibition of the
enzyme in the assembled complex, sequentially mix ODN1TEM1- β-lactamase, ssDNA template (with a target recognition sequence length of 0, 10, 20, 30, 40, or 50 nucleotides)
and ODN2-BLIP in a 1:1.2:2 ratio at low micromolar concentrations in complexation buffer and incubate for 30 minutes at
room temperature.
4. Mix the hybridized samples prepared in step 2 with an equal
volume of 2× sample buffer and load it on a semi-native 12%
SDS-PAGE gel prepared in step 1. Run the gel for 15 min at
90 V followed by 1 h at 125 V.
5. Remove the gel from the glass plates and wash with dH 2 O for
5 min. Subsequently stain the gel with Coomassie blue for
30 min. Then destain the gel with dH 2 O until clear bands
appear and image the gel (Fig. 4).
1. Mix ODN1-TEM1-β-lactamase, ODN2-BLIP, and ssDNA
template (with target recognition sequences of 0, 10, 20, 30,
40, and 50 nucleotides) in PBS
+
at low micromolar concentrations in a ratio of 1:2:1.2 and incubate for 30 min at room
temperature.
2. Dilute the mixtures in PBS
+
to a final concentration of 50 nM
ODN1-TEM1-β-lactamase.
3. In a 96-well plate, mix 4 μL of the diluted complexes with
152 μL PBS
+
and 24 μL target oligonucleotides (from 100 nM
stock solution in PBS
+
) and incubate for 1 h at room
temperature.
4. Add 20 μL nitrocefin (from 500 μM stock solution in PBS)
yielding a final concentration of 1 nM DNA-directed enzymeinhibitor complex, 12 nM target oligonucleotide, and 50 μM
nitrocefin.
5. Directly place the plate in the platereader and proceed with
measuring the optical absorbance in time of the formed product
at 486 nm.
3.4 Semi-native
SDS-PAGE
of Reconstituted
DNA-Directed EnzymeInhibitor Complex
3.5 Enzyme Activity
Assay of Reconstituted
DNA-Directed EnzymeInhibitor Complex
Wouter Engelen and Maarten Merkx