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<div class="entry"><big><big><big><big><span
<div class="entry"><big><big><big><big><span
  style="color: black; font-weight: bold;">Functionalized ZFPs</span></big></big></big></big><br>
  style="color: black; font-weight: bold;">Functionalized ZFPs</span></big></big></big></big><br>
<br>
<br><br>
<span style="font-family: Arial;">
<span style="font-family: Arial;">
Protein domains exhibit a wide variety of physicochemical properties
Protein domains exhibit a wide variety of physicochemical properties and represent a rich source for delivery of the specific function to a defined position on DNA origami. In order to demonstrate the proof of the concept for spatial addressing of protein functions to a DNA origami, we considered using restriction enzymes, proteases, biosynthetic enzymes, proteins with optical properties, motor proteins etc.<br>
and represent an important pool for delivering the specific function to
a defined position on DNA origami. In order to demonstrate the proof of
the concept for spatial addressing of protein functions on a DNA
origami, we considered using restriction enzymes, proteases,
biosynthetic enzymes, proteins with optical properties, motor proteins
etc.<br>
&nbsp;</span><br style="font-family: Arial;">
&nbsp;</span><br style="font-family: Arial;">
<span style="font-family: Arial;">Finally we decided to explore the performance of BRET - bioluminescence energy transfer on the DNA origami. BRET is a biophysical phenomenon in which energy is non-radiatively transferred from the donor (Renilla luciferase in our case) to the acceptor (mCitrine variant of the yellow fluorescent protein) when both molecules are in close proximity (within 10 - 100 Å range). Renilla luciferase (RLuc) catalyzes the reaction of oxidative decarboxylation converting its substrate coelenterazine to coelenteramide by the simultaneous emission of light with a maximum emission peak around 475 nm. The energy of photons is high enough to excite the yellow fluorescent protein (in case it is bound within the required distance from the Renilla luciferase) leading to fluorescence with an emission maximum of 529 nm. Upon successful implementation of binding of BRET pair to DNA origami, the system could be expanded to four partners by splitting each of Renilla luciferase and mCitrine into 2 segments, where BRET would depend on the proximity of four sites (two pairs). Because of the distance-dependent signal efficiency, BRET could be employed as a nanomolecular ruler or as a logical operator for <a
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">biosensors</a> as outlined in the Applications section.
</span><br><br>
<table
<table
  style="margin: 3px 0px 20px 20px; width: 444px; height: 50px; float: right;"
  style="margin: 3px 0px 20px 20px; width: 100%; height: 50px; float: right;"
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   <tbody>
   <tbody>
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       <td style="text-align: center;"><img
       <td style="text-align: center;"><img
  style="padding: 0pt 0pt 5px; width: 460px; height: 345px;"
  style="padding: 0pt 0pt 5px; width: 890px; height: 238px;"
  alt="This is picture of origami rose" title="Origami rose"
  alt="" title=""
  src="http://upload.wikimedia.org/wikipedia/commons/thumb/6/61/Rosa_de_papel.jpg/1024px-Rosa_de_papel.jpg"></td>
  src="http://openwetware.org/images/9/9a/BRET_final.png"></td>
     </tr>
     </tr>
     <tr style="font-family: Arial; font-weight: bold;">
     <tr style="font-family: Arial; font-weight: bold;">
       <td style="text-align: justify;">Figure.<span
       <td style="text-align: justify;">Figure 18: Functionalized ZFPs decorating DNA origami plane. <span
  style="font-weight: normal;"> Slika Modela BRET fuzij ali
  style="font-weight: normal;"> (a) Molecular models of BRET triple fusion proteins, namely MBP-RLuc-2C7 and MBP-mCitrine-AZPA4, are presented. BRET is observed after Renilla luciferase catalyses oxidative decarboxylation of its substrate coelenterazine to coelenteramide which in terms emits light at ~ 475 nm and excites mCitrine when in close proximity (1 - 10 nm) (b) DNA origami plane decorated with BRET partner fusions.</span></td>
shema delovanja BRETa (ni v sekciji metod)</span></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
<span style="font-family: Arial;">Finally we decided to
<span style="font-family: Arial;">We first prepared constructs for BRET protein chimeras in the form of RLuc-2C7 and YFP-AZPA4. Unfortunately, it turned out the two proteins were expressed exclusively in the insoluble fraction of the cell lysate (Figure 19), which means that we would need an efficient refolding procedure to prepare functional proteins for binding to DNA origami. We tried several isolation and refolding procedures with little success. </span><br style="font-family: Arial;">
explore the performance of BRET - bioluminescence energy transfer on
the DNA origami. BRET is a biophysical phenomenon in which energy is
non-radiatively transferred from the donor (Renilla luciferase in our
case) to the acceptor (mCitrine variant of the yellow fluorescent
protein) when both molecules are bound in close proximity (within 10 -
100Å range). Renilla luciferase (RLuc) catalyzes the reaction of
oxidative decarboxylation converting its substrate coelenterazine to
coelenteramide which emits light with a maximium emission peak at
around 475 nm. The energy of photons is high enough to excite yellow
fluorescent protein (in case it is bound within the required distance
away from Renilla luciferase) leading to fluorescence with an emission
maximum of 529 nm. Upon successful implementation of a BRET pair
binding to DNA origami, the system could be expanded to four partners
by splitting Renilla luciferase and mCitrine in 2 segments, where BRET
would depend on the proximity of four sites (two pairs). Because of the
distance-dependent signal efficiency, BRET could be employed as a
nanomolecular ruler or as a logical operator for <a
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">biosensors</a>
as outlined
in the Applications section.
</span><br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">We first prepared
constructs for BRET protein chimeras in the form of RLuc-2C7 and
YFP-AZPA4. Unfortunately, it turned out the two proteins were expressed
exclusively in the insoluble fraction of the cell lysate, meaning they
were aggregated and would need refolding for binding to DNA origami.
Several isolation and refolding attempts were tried with little
success. </span><br style="font-family: Arial;">
<br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">We tried to optimize the
<span style="font-family: Arial;">Next, we tried to optimize the recombinant protein production conditions in order to purify the produced protein fusions in the native form. A well-known strategy for enhancing solubility and proper folding of larger protein fusions during their production is to lower the temperature as well as the concentration of the inducer IPTG allowing the protein to fold correctly and decrease the formation of protein aggregates <em>in vivo</em>. We achieved partial expression of soluble YFP-AZPA4 (Figure 20), with no improvement on solubilization of RLUC-2C7 fusion.</span><br style="font-family: Arial;">
production conditions in order to purify the produced protein fusions
in the native form. A well-known strategy for enhancing solubility and
proper folding of larger protein fusions during their production is to
lower the temperature as well as the concentration of the inducer IPTG
giving the protein time to fold correctly and decrease the formation of
protein aggregates in vivo. We achieved partial expression of soluble
YFP-AZPA4 (Figure 2), with no improvement on solubilization of RLUC-2C7
fusion.</span><br style="font-family: Arial;">
<br>
<br>
<table style="text-align: left; width: 100%;" border="0"
<table style="text-align: left; width: 100%;" border="0"
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       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-weight: bold;">Figure
  style="font-weight: bold;">Figure
1. ZFP-luciferase and -YFP fusions are produced in bacteria in an
19. ZFP-luciferase and ZFP-YFP fusions are produced in bacteria in an
insoluble form.</span> Figure shows Western blot after production
insoluble form.</span> Figure shows Western blot after production of fusion proteins. Arrows to the left indicate the position of bands representing BRET fusions without MBP solubility tag present in an insoluble form (IBs - inclusion bodies). Grey arrow depicts the position of YFP-AZPA4 (Mr of 48 kDa) and black arrow the position of RLuc-2C7 (Mr of 59 kDa). Production conditions: 2x YT medium supplemented with 0.1 mM ZnCl<sub>2</sub> and 100 mg/L antibiotic kanamycin / 37 °C / 180 rpm / ~7 h induction with 2 mM IPTG.</td>
of fusion
proteins intended for the BRET assay. Arrows to the left indicate the
position of bands representing BRET fusions without MBP solubility tag
present in an insoluble form (IBs - inclusion bodies). Grey arrow
depicts the position of YFP-AZPA4 (Mr of 48.4 kDa) and black arrow the
position of RLuc-2C7 (Mr of 59.5 kDa). Production conditions: 2x YT
medium supplemented with 0.1 mM ZnCl2 and 100 mg/L antibiotic kanamycin
/ 37 °C / 180 rpm / ~7 h induction with 2 mM IPTG. </td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-weight: bold;">Figure
  style="font-weight: bold;">Figure
2. Production of soluble YFP-AZPA4 fusion by modification of production
20. Production of low amount of soluble YFP-AZPA4 fusion by modification of production conditions.</span> Western blot after production of YFP-AZPA4
conditions.</span> Western blot after production of YFP-AZPA4
with altered fermentation conditions (LB medium supplemented with 0.5 mM ZnCl<sub>2</sub> and 100 mg/L antibiotic kanamycin / 24 °C / 160 rpm / ~24 h induction with
with altered
0.5 mM IPTG). This made YFP-AZPA4 protein (Mr of 48 kDa) observable
fermentation conditions (LB medium supplemented with 0.5 mM ZnCl2 and
100 mg/L antibiotic kanamycin / 24 °C / 160 rpm / ~24 h induction with
0.5 mM IPTG). This made YFP-AZPA4 protein (Mr of 48.3 kDa) observable
in the soluble form (depicted by the arrow to the right), but not to
in the soluble form (depicted by the arrow to the right), but not to
sufficient extent to isolate it. </td>
sufficient extent to isolate it. </td>
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</table>
</table>
<br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">Since we have already
<span style="font-family: Arial;">Since we have already observed the beneficial effect of enhanced solubility by attaching MBP and/or GST domains to the N-terminal end of zinc finger proteins (section <a
observed a beneficial effect of enhanced solubility by the attachment
of MBP and/or GST domains to the N-terminal end of zinc finger proteins
(section <a
  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">Soluble
  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">Soluble
ZFPs</a>), MBP domain was added to the N-termini of both
ZFPs</a>), MBP domain was added to the N-termini of both BRET fusion proteins, creating triple fusion proteins in the following form: MBP-RLuc-2C7 and MBP-YFP-AZPA4, aiming to increase their solubility and facilitate the isolation of functional proteins under native conditions.</span><br style="font-family: Arial;">
BRET fusion proteins, creating triple fusion proteins in the following
form: MBP-RLuc-2C7 and MBP-YFP-AZPA4 with an aim to increase their
solubility and foster the isolation of functional proteins under native
conditions.</span><br style="font-family: Arial;">
<br>
<br>
<span style="font-family: Arial;">We anticipated that
<span style="font-family: Arial;">We anticipated that decreasing the temperature during protein production would lead to further increase of final protein titers but ultimately we observed the solubility of both triple chimeras was comparable at 30 °C and 37 °C as can be inferred from the results below. We used the following conditions for the production: 2x YT medium supplemented with 10 g/L glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl<sub>2</sub> / 30 °C or 37 °C / 160 rpm / ~5 or 7 h induction with 1 mM IPTG.</span><br><br>
lowering the temperature during the protein production would lead to
further increase of the final protein titers but ultimately we observed
the solubility of both triple chimeras was comparable at 30 °C and 37
°C as can be inferred from the results below. We used the following
conditions for the production: 2x YT medium supplemented with 10 g/L
glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl2 / 30 °C or 37
°C / 160rpm / ~5 or 7 h induction with 1 mM IPTG.</span><br>
<span style="font-family: Arial;"></span><br>
<span style="font-family: Arial;"></span><br>
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     <tr>
       <td style="text-align: left;"><img
       <td style="text-align: left;"><img
  style="margin-bottom: 10px; width: 890px; height: 430px;" alt=""
  style="margin-bottom: 10px; width: 593px; height: 287px;" alt=""
  src="http://openwetware.org/images/0/01/BRETfusionswithsolubilityTagproductionCBB.png"></td>
  src="http://openwetware.org/images/0/01/BRETfusionswithsolubilityTagproductionCBB.png"></td>
     </tr>
     </tr>
     <tr>
     <tr>
       <td style="text-align: justify;" valign="undefined"><span
       <td style="text-align: justify;" valign="undefined"><span
  style="font-weight: bold;">Figure 3. ZFP-BRET-MBP fusions
  style="font-weight: bold;">Figure 21. ZFP-BRET-MBP fusions are produced in soluble form.</span> Bacterial lysate fractions after production of BRET fusions with MBP domain were analyzed with Coomassie Brilliant Blue stained SDS PAGE analysis. Grey arrow to the right indicates the position of MBP-YFP-AZPA4 (Mr of 90 kDa) and black arrow the position of MBP-RLuc-2C7 (Mr of 101 kDa). Addition of MBP solubility tag promoted the presence of both protein chimeras in supernatant (SN). Both proteins were partially present in inclusion bodies (IBs) which was the case with all other ZFP chimeras as well.</td>
are produced in soluble form.</span> Coomassie Brilliant Blue
stain of bacterial lysate fractions after production of BRET fusions
with MBP domain. Grey arrow to the right indicates the approximate
position of MBP-YFP-AZPA4 (Mr of 90.4 kDa) and black arrow the position
of MBP-RLuc-2C7 (Mr of 101.5 kDa). As anticipated, the addition of MBP
solubility tag promoted the presence of both protein chimeras in
supernatant (SN). Both proteins were partially present in inclusion
bodies (IBs) which was the case with all other ZFP chimeras as well.</td>
     </tr>
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   </tbody>
   </tbody>
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       <td style="text-align: left;"><img
       <td style="text-align: left;"><img
  style="margin-bottom: 10px; width: 882px; height: 499px;" alt=""
  style="margin-bottom: 10px; width: 558px; height: 287px;" alt=""
  src="http://openwetware.org/images/0/0e/BRETfusionswithsolubilitytagproductionWB.png"></td>
  src="http://openwetware.org/images/0/0e/BRETfusionswithsolubilitytagproductionWB.png"></td>
     </tr>
     </tr>
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       <td style="text-align: justify;" valign="undefined"><span
       <td style="text-align: justify;" valign="undefined"><span
  style="font-weight: bold;">Figure 4. ZFP-BRET-MBP fusions
  style="font-weight: bold;">Figure 22. ZFP-BRET-MBP fusions are produced in soluble form. </span>Western Blot after production of BRET fusions with MBP tag. Grey arrow to the right indicates the expected position of MBP-YFP-AZPA4 (Mr of 90 kDa) and black arrow the position of MBP-RLuc-2C7 (Mr of 101 kDa).<br><br></td>
are produced in soluble form. </span>Western Blot after
production of BRET fusions with MBP tag. Grey arrow to the right
indicates the expected position of MBP-YFP-AZPA4 (Mr of 90.4 kDa) and
black arrow the position of MBP-RLuc-2C7 (Mr of 101.5 kDa).</td>
     </tr>
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   </tbody>
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       <td style="text-align: left;"><img
       <td style="text-align: left;"><img
  style="margin-right: 20px; width: 350px; height: 536px;" alt=""
  style="margin-right: 20px; width: 200px; height: 306px;" alt=""
  src="http://openwetware.org/images/6/63/BRETfusionsisolationCBB.png"></td>
  src="http://openwetware.org/images/6/63/BRETfusionsisolationCBB.png"></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-weight: bold;">Figure 5. Isolation of BRET
  style="font-weight: bold;">Figure 23. Isolation of BRET fusions with MBP tag using chelating chromatography.</span> Coomassie Brilliant Blue (CBB) stained SDS PAGE analysis of isolated fractions. Isolation of both BRET triple fusion proteins (MBP-YFP-AZPA4, Mr of 90 kDa and MBP-RLuc-2C7, Mr of 101 kDa) resulted in a single protein band observed after staining the SDS-PAGE gel with CBB. The yield of MBP-YFP-AZPA4 was approximately 10-times higher compared to MBP-RLuc-2C7. </td>
fusions with MBP tag using chelating chromatography.</span>
Coomassie Brilliant Blue (CBB) stain of isolated fractions. Isolation
of both BRET triple fusion proteins (MBP-YFP-AZPA4, Mr of 90.4 kDa and
MBP-RLuc-2C7, Mr of 101.5 kDa) resulted in a single protein band
observed after staining the SDS-PAGE gel with CBB. However, the
isolation efficiency of MBP-YFP-AZPA4 was approximately 10-times higher
compared to MBP-RLuc-2C7 as observed when determining proteins'
concentrations after isolation. </td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
<span style="font-family: Arial;"><br>
<span style="font-family: Arial;"><br>
After protein isolation various functional assays were performed to
After protein isolation functional assays were performed to analyze the effect of added protein domains at both N- and C- terminus of yellow fluorescent protein (mCitrine) and Renilla luciferase (RLuc). We determined the fluorescence of YFP fusions and decarboxylation enzymatic activity on the luciferase's substrate coelenterazine leading to light emission in case of luciferase.</span><br>
analyse the effect of added protein domains at both N- and C- terminus
of yellow fluorescent protein (mCitrine) and Renilla luciferase (RLuc)
We determined the fluorescence of YFP fusions and decarboxylation
enzymatic activity on the luciferase's substrate coelenterazine leading
to light emission in case of luciferase.</span><br>
<br>
<br>
<br>
<br>
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       <td style="text-align: left;"><img
       <td style="text-align: left;"><img
  style="margin-right: 20px; width: 500px; height: 223px;" alt=""
  style="margin-right: 20px; width: 450px; height: 223px;" alt=""
  src="http://openwetware.org/images/7/72/MBPYFPAZPA4fluorescentspectrum.png"></td>
  src="http://openwetware.org/images/7/72/MBPYFPAZPA4fluorescentspectrum.png"></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-weight: bold;">Figure 6. Flurescence emission
  style="font-weight: bold;">Figure 24. Fluorescence emission spectrum of MBP-YFP-AZPA4 fusion. </span>
spectrum of MBP-YFP-AZPA4 fusion. </span>
The fluorescence emission spectrum of MBP-YFP-AZPA4 chimera was acquired on a PerkinElmer LS55 Luminescence Spectrometer. Protein sample was excited at 485 nm and emission spectra were obtained in the 500 to 600 nm window with a scanning speed of 100 nm/min. Red line represents blank control (lysis buffer), green line is a negative control (2C7-MBP-6F6 supernatant) and blue line shows the fluorescence emission spectrum of MBP-YFP-AZPA4 chimera.</td>
Using PerkinElmer LS55 Luminescence Spectrometer the fluorescence
emission spectrum of MBP-YFP-AZPA4 chimera was acquired. 130 μl of the
supernatant was transferred to Hellma QS 3 mm quartz cuvette and
excited at 485 nm. Emission spectrum was obtained within the 500 to 600
nm emission window with a scanning speed of 100 nm/min. Excitation and
emission slits were adjusted to 5 nm. Red line represents blank control
(lysis buffer), green line is a negative control (2C7-MBP-6F6
supernatant) and blue line shows the fluorescence emission spectrum of
MBP-YFP-AZPA4 chimera.</td>
     </tr>
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   </tbody>
   </tbody>
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       <td valign="undefined"><span
       <td valign="undefined"><span
  style="font-weight: bold;">Figure 7. Visual characterization
  style="font-weight: bold;">Figure 25. Visual characterization of the isolated MBP-YFP-AZPA4 chimera under UV light. </span>
of the isolated MBP-YFP-AZPA4 chimera under UV light. </span>
Left: centrifuge tubes with isolated MBP-YFPA-AZPA4 (50, 100 and 250 mM imidazole elution fractions) were illuminated with UV light. Negative control (isolated AZPA4-MBP-6F6) was added to the left of the three fluorescent samples to demonstrate the difference in optical properties of isolated proteins. Right: 200 μl of 100mM imidazole elution fraction of MBP-YFP-AZPA4 was pipetted into Corning Costar 96-well white microtiter plate with transparent bottom to yield a pattern of "YFP". The plate was analyzed in DNA Bio-Imaging Systems box by illumination with the UV light.</td>
Left: centrifuge tubes with isolated MBP-YFPA-AZPA4 (50, 100 and 250 mM
imidazole elution fractions) were illuminated with UV light. Negative
control (isolated AZPA4-MBP-6F6) was added to the left of the three
fluorescent samples to signify the difference in proteins optical
properties. Right: 200 μl of 100mM imidazole elution fraction of
MBP-YFP-AZPA4 was pippeted into Corning Costar 96-well white microtiter
plate with transparent bottom to yield a pattern of "YFP". The plate
was put into DNA Bio-Imaging Systems box and illuminated with the UV
light.</td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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</span></big></big><span
</span></big></big><span
  style="font-family: Arial;"><br>
  style="font-family: Arial;"><br>
<table style="text-align: left; width: 80%;" border="0"
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  src="http://openwetware.org/images/2/21/MBPRLuc2C7biolum.png"></td>
  src="http://openwetware.org/images/2/21/MBPRLuc2C7biolum.png"></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-weight: bold;">Figure 8. Bioluminescence
  style="font-weight: bold;">Figure 26. MBP-RLuc-2C7 ZFP chimera exhibits bioluminsecence. </span>
measurement for MBP-RLuc-2C7 chimera. </span>
40 μl of MBP-RLuc-2C7 supernatant (non-diluted, diluted 2 or 5-fold) was transferred to Corning Costar 96-well white microtiter plate. The bioluminescence assay was performed on a Berthold's ORION II Microplate Luminometer.</td>
40 μl of MBP-RLuc-2C7 supernatant (non-diluted, diluted 2 or 5-fold)
was transferred to Corning Costar 96-well white microtiter plate. The
bioluminescence assay was performed as follows using Berthold's ORION
II Microplate Luminometer. After putting the microplate into
luminometer 100 μl of 8 μM substrate coelenterazine h (Synchem) was
injected into each well followed by photon emission as a result of
luminescence, which was collected by the instrument for 1 sec and
converted to RLU (relative luminescence units).</td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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       <td><span style="font-family: Arial;"><img
       <td><span style="font-family: Arial;"><img
  style="margin-right: 20px; width: 500px; height: 164px;" alt=""
  style="margin-right: 20px; width: 780px; height: 255px;" alt=""
  src="http://openwetware.org/images/8/83/MBPRLuc2C7visual.png"></span></td>
  src="http://openwetware.org/images/8/83/MBPRLuc2C7visual.png"></span></td>
     </tr>
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  style="font-weight: bold;"></span><span
  style="font-weight: bold;"></span><span
  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">Figure 9. Visual characterization
  style="font-weight: bold;">Figure 27. Visual characterization of the activity of MBP-RLuc-2C7 chimera. </span>
of the activity of MBP-RLuc-2C7 chimera. </span>
Left: 100 μl of MBP-RLuc-2C7 supernatant was transferred to Perkin Elmer OptiPlateTM 96-well white plate in a pattern giving rise to the word "Luc". Right: 100 μl of the coelenterazine h substrate was added to the protein sample prepared in the same way as for the bioluminescence assay. Luminescence was detected in Syngene G:Box for 5 sec. <br><br></span></td>
Left: 100 μl of MBP-RLuc-2C7 supernatant was transferred to Perkin
Elmer OptiPlateTM 96-well white plate in a pattern giving rise to the
word "Luc". Right: After adding 100 μl of the coelenterazine h
substrate solution prepared in the same way as for the bioluminescence
assay with ORION II, the luminescence was collected in Syngene G:Box
for 5 sec. Since Renilla luciferase exhibits fast reaction kinetics
under these buffer conditions, some spots are brighter than others
which is due to the fact that the substrate was not added into all
microtiter wells simultaneously.</span></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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<br>
<br>
</span><big><big><span
</span><big><big><span
  style="color: black; font-weight: bold;">BRET on DNA target</span></big></big><span
  style="color: black; font-weight: bold;">BRET effect to detect neighboring binding sites on DNA target</span></big></big><span
  style="font-family: Arial;"><br>
  style="font-family: Arial;"><br>
<br>
<br>
Line 800: Line 674:
     <tr align="center">
     <tr align="center">
       <td><span style="font-family: Arial;"><img
       <td><span style="font-family: Arial;"><img
  style="margin-right: 20px; width: 400px; height: 68px;" alt=""
  style="margin-right: 20px; width: 780px; height: 133px;" alt=""
  src="http://openwetware.org/images/3/32/BRETDNAtarget.png"></span></td>
  src="http://openwetware.org/images/3/32/BRETDNAtarget.png"></span></td>
     </tr>
     </tr>
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  style="font-weight: bold;"></span><span
  style="font-weight: bold;"></span><span
  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">Figure 10: Sequence of the BRET
  style="font-weight: bold;">Figure 28: Sequence of the DNA target designed for BRET experiment. </span>
DNA target. </span>
43 bp DNA target used to measure the BRET effect was designed as shown above. 2C7 binding sequence is colored blue and AZPA4 binding site in green. Spacer of 2 bp was selected to separate the binding sites and a clamp of the same length at both 5' and 3' end.<br><br></span></td>
43 bp DNA target used to measure the BRET effect was designed as shown
above. Blue coloured is the 2C7 binding sequence and depicted in green
is AZPA4 binding site. 2 bp long spacer was selected to separate the
binding sites and flank of the same length at both 5' and 3' ends.</span></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
Line 825: Line 695:
  src="http://openwetware.org/images/b/b8/BRETgraphDNA.png"></td>
  src="http://openwetware.org/images/b/b8/BRETgraphDNA.png"></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-weight: bold;">Figure 11: BRET assay of the
  style="font-weight: bold;">Figure 29: BRET resulting from the addition of DNA target sequence to chimeric ZFP fusions. </span>
mixture of chimeric ZFP fusions. </span>
mCitrine emission was normalized to the 535 nm emission of the MBP-RLuc-2C7. Since the emission spectra of RLuc and mCitrine overlap, 535 nm emission is observed for 100 nM MBP-RLuc-2C7 as well. Addition of 50 nM DNA target increased mCitrine emission indicating proximal binding of chimeric proteins to the DNA target.</td>
mCitrine emission was normalized to the 535 nm emission of the
MBP-RLuc-2C7 without other protein or DNA components. Since the
emission spectra of RLuc and mCitrine overlap, 535 nm emission peak is
observed for 100nM MBP-RLuc-2C7 alone in the sample as well. When both
BRET partners in 100 nM concentration are present and no DNA 535 nm
emission peak increases possibly due to the random collisions between
the molecules in the system. However, the addition of 50 nM DNA target
furtherly inceased mCitrine emission indicating the proximal binding of
both fusions to the DNA target.</td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
<br>
<br>
We demonstrated the design, production, purification and
We demonstrated the design, production, purification and characterization of the two protein elements required for the reconstitution of the BRET sensor on a DNA origami. Initial experiments support the functional BRET, however the overlapping Renilla luciferase and mCitrine emission spectra suggest that selection of other BRET partners might improve the signal to background ratio, e.g. replace the Rluc-YFP pair with RLuc8-GFP2 BRET pair as a reasonable choice (De, 2007). Sensitivity of the BRET experiment on DNA origami could be improved by binding several neighboring BRET pairs on a single DNA origami rectangle.<br>
characterization of the two protein elements required for the
reconstitution of the BRET sensor on a DNA origami. Initial experiments
support the functional BRET, however the overlapping Renilla luciferase
and mCitrine emission spectra suggest that selection of other BRET
partners might improve the signal to background ratio, e.g. RLuc8-GFP2
BRET pair as a reasonable choice (De, 2007). Sensitivity of the BRET
effect on DNA origami could be improved by mounting several neighboring
BRET pairs on a single DNA origami rectangle.<br>
<br>
<br>
</span>
</span>

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</ul> <!-- End PureCSSMenu.com MENU --> </div> </div> </div> <!-- end #header --> <div id="page" class="container"> <div id="content"> <div class="post"> <div class="entry"><big><big><big><big><span 

style="color: black; font-weight: bold;">Functionalized ZFPs</span></big></big></big></big><br>

<br><br> <span style="font-family: Arial;"> Protein domains exhibit a wide variety of physicochemical properties and represent a rich source for delivery of the specific function to a defined position on DNA origami. In order to demonstrate the proof of the concept for spatial addressing of protein functions to a DNA origami, we considered using restriction enzymes, proteases, biosynthetic enzymes, proteins with optical properties, motor proteins etc.<br> &nbsp;</span><br style="font-family: Arial;">

<span style="font-family: Arial;">Finally we decided to explore the performance of BRET - bioluminescence energy transfer on the DNA origami. BRET is a biophysical phenomenon in which energy is non-radiatively transferred from the donor (Renilla luciferase in our case) to the acceptor (mCitrine variant of the yellow fluorescent protein) when both molecules are in close proximity (within 10 - 100 Å range). Renilla luciferase (RLuc) catalyzes the reaction of oxidative decarboxylation converting its substrate coelenterazine to coelenteramide by the simultaneous emission of light with a maximum emission peak around 475 nm. The energy of photons is high enough to excite the yellow fluorescent protein (in case it is bound within the required distance from the Renilla luciferase) leading to fluorescence with an emission maximum of 529 nm. Upon successful implementation of binding of BRET pair to DNA origami, the system could be expanded to four partners by splitting each of Renilla luciferase and mCitrine into 2 segments, where BRET would depend on the proximity of four sites (two pairs). Because of the distance-dependent signal efficiency, BRET could be employed as a nanomolecular ruler or as a logical operator for <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">biosensors</a> as outlined in the Applications section.

</span><br><br> <table 

style="margin: 3px 0px 20px 20px; width: 100%; height: 50px; float: right;"
border="0" cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: center;"><img
style="padding: 0pt 0pt 5px; width: 890px; height: 238px;"
alt="" title=""
src="http://openwetware.org/images/9/9a/BRET_final.png"></td>
   </tr>
   <tr style="font-family: Arial; font-weight: bold;">
     <td style="text-align: justify;">Figure 18: Functionalized ZFPs decorating DNA origami plane. <span
style="font-weight: normal;"> (a) Molecular models of BRET triple fusion proteins, namely MBP-RLuc-2C7 and MBP-mCitrine-AZPA4, are presented. BRET is observed after Renilla luciferase catalyses oxidative decarboxylation of its substrate coelenterazine to coelenteramide which in terms emits light at ~ 475 nm and excites mCitrine when in close proximity (1 - 10 nm) (b) DNA origami plane decorated with BRET partner fusions.</span></td>
   </tr>
 </tbody>

</table> <span style="font-family: Arial;">We first prepared constructs for BRET protein chimeras in the form of RLuc-2C7 and YFP-AZPA4. Unfortunately, it turned out the two proteins were expressed exclusively in the insoluble fraction of the cell lysate (Figure 19), which means that we would need an efficient refolding procedure to prepare functional proteins for binding to DNA origami. We tried several isolation and refolding procedures with little success. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">Next, we tried to optimize the recombinant protein production conditions in order to purify the produced protein fusions in the native form. A well-known strategy for enhancing solubility and proper folding of larger protein fusions during their production is to lower the temperature as well as the concentration of the inducer IPTG allowing the protein to fold correctly and decrease the formation of protein aggregates <em>in vivo</em>. We achieved partial expression of soluble YFP-AZPA4 (Figure 20), with no improvement on solubilization of RLUC-2C7 fusion.</span><br style="font-family: Arial;"> <br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="8">
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   <tr>
     <td style="text-align: center;"><img
style="width: 276px; height: 374px;" alt=""
src="http://openwetware.org/images/1/1f/InsolubleBRETfusionsWBproduction.png"></td>
     <td style="text-align: center;"><img alt=""
src="http://openwetware.org/images/6/66/MBPYFPAZPA4alteredcond.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure

19. ZFP-luciferase and ZFP-YFP fusions are produced in bacteria in an insoluble form.</span> Figure shows Western blot after production of fusion proteins. Arrows to the left indicate the position of bands representing BRET fusions without MBP solubility tag present in an insoluble form (IBs - inclusion bodies). Grey arrow depicts the position of YFP-AZPA4 (Mr of 48 kDa) and black arrow the position of RLuc-2C7 (Mr of 59 kDa). Production conditions: 2x YT medium supplemented with 0.1 mM ZnCl<sub>2</sub> and 100 mg/L antibiotic kanamycin / 37 °C / 180 rpm / ~7 h induction with 2 mM IPTG.</td> 

     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure

20. Production of low amount of soluble YFP-AZPA4 fusion by modification of production conditions.</span> Western blot after production of YFP-AZPA4 with altered fermentation conditions (LB medium supplemented with 0.5 mM ZnCl<sub>2</sub> and 100 mg/L antibiotic kanamycin / 24 °C / 160 rpm / ~24 h induction with 0.5 mM IPTG). This made YFP-AZPA4 protein (Mr of 48 kDa) observable in the soluble form (depicted by the arrow to the right), but not to sufficient extent to isolate it. </td> 

   </tr>
 </tbody>

</table> <br style="font-family: Arial;"> <span style="font-family: Arial;">Since we have already observed the beneficial effect of enhanced solubility by attaching MBP and/or GST domains to the N-terminal end of zinc finger proteins (section <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">Soluble

ZFPs</a>), MBP domain was added to the N-termini of both BRET fusion proteins, creating triple fusion proteins in the following form: MBP-RLuc-2C7 and MBP-YFP-AZPA4, aiming to increase their solubility and facilitate the isolation of functional proteins under native conditions.</span><br style="font-family: Arial;"> <br> <span style="font-family: Arial;">We anticipated that decreasing the temperature during protein production would lead to further increase of final protein titers but ultimately we observed the solubility of both triple chimeras was comparable at 30 °C and 37 °C as can be inferred from the results below. We used the following conditions for the production: 2x YT medium supplemented with 10 g/L glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl<sub>2</sub> / 30 °C or 37 °C / 160 rpm / ~5 or 7 h induction with 1 mM IPTG.</span><br><br> <span style="font-family: Arial;"></span><br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
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     <td style="text-align: left;"><img
style="margin-bottom: 10px; width: 593px; height: 287px;" alt=""
src="http://openwetware.org/images/0/01/BRETfusionswithsolubilityTagproductionCBB.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify;" valign="undefined"><span
style="font-weight: bold;">Figure 21. ZFP-BRET-MBP fusions are produced in soluble form.</span> Bacterial lysate fractions after production of BRET fusions with MBP domain were analyzed with Coomassie Brilliant Blue stained SDS PAGE analysis. Grey arrow to the right indicates the position of MBP-YFP-AZPA4 (Mr of 90 kDa) and black arrow the position of MBP-RLuc-2C7 (Mr of 101 kDa). Addition of MBP solubility tag promoted the presence of both protein chimeras in supernatant (SN). Both proteins were partially present in inclusion bodies (IBs) which was the case with all other ZFP chimeras as well.</td>
   </tr>
 </tbody>

</table> <br> <br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
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     <td style="text-align: left;"><img
style="margin-bottom: 10px; width: 558px; height: 287px;" alt=""
src="http://openwetware.org/images/0/0e/BRETfusionswithsolubilitytagproductionWB.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify;" valign="undefined"><span
style="font-weight: bold;">Figure 22. ZFP-BRET-MBP fusions are produced in soluble form. </span>Western Blot after production of BRET fusions with MBP tag. Grey arrow to the right indicates the expected position of MBP-YFP-AZPA4 (Mr of 90 kDa) and black arrow the position of MBP-RLuc-2C7 (Mr of 101 kDa).<br><br></td>
   </tr>
 </tbody>

</table> <span style="font-family: Arial;"><br> </span> <table style="text-align: left; width: 75%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 200px; height: 306px;" alt=""
src="http://openwetware.org/images/6/63/BRETfusionsisolationCBB.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 23. Isolation of BRET fusions with MBP tag using chelating chromatography.</span> Coomassie Brilliant Blue (CBB) stained SDS PAGE analysis of isolated fractions. Isolation of both BRET triple fusion proteins (MBP-YFP-AZPA4, Mr of 90 kDa and MBP-RLuc-2C7, Mr of 101 kDa) resulted in a single protein band observed after staining the SDS-PAGE gel with CBB. The yield of MBP-YFP-AZPA4 was approximately 10-times higher compared to MBP-RLuc-2C7.  </td>
   </tr>
 </tbody>

</table> <span style="font-family: Arial;"><br> After protein isolation functional assays were performed to analyze the effect of added protein domains at both N- and C- terminus of yellow fluorescent protein (mCitrine) and Renilla luciferase (RLuc). We determined the fluorescence of YFP fusions and decarboxylation enzymatic activity on the luciferase's substrate coelenterazine leading to light emission in case of luciferase.</span><br> <br> <br> <big><big><span style="color: black; font-weight: bold;">Characterization of MBP-YFP-AZPA4</span></big></big><br 

style="font-family: Arial;">

<br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 450px; height: 223px;" alt=""
src="http://openwetware.org/images/7/72/MBPYFPAZPA4fluorescentspectrum.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 24. Fluorescence emission spectrum of MBP-YFP-AZPA4 fusion. </span>

The fluorescence emission spectrum of MBP-YFP-AZPA4 chimera was acquired on a PerkinElmer LS55 Luminescence Spectrometer. Protein sample was excited at 485 nm and emission spectra were obtained in the 500 to 600 nm window with a scanning speed of 100 nm/min. Red line represents blank control (lysis buffer), green line is a negative control (2C7-MBP-6F6 supernatant) and blue line shows the fluorescence emission spectrum of MBP-YFP-AZPA4 chimera.</td> 

   </tr>
 </tbody>

</table> <br> <br> <table style="text-align: left; width: 80%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr align="center">
     <td><img
style="margin-bottom: 10px; width: 750px; height: 254px;" alt=""
src="http://openwetware.org/images/2/23/MBPYFPAZPA4visual.png"></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-weight: bold;">Figure 25. Visual characterization of the isolated MBP-YFP-AZPA4 chimera under UV light. </span>

Left: centrifuge tubes with isolated MBP-YFPA-AZPA4 (50, 100 and 250 mM imidazole elution fractions) were illuminated with UV light. Negative control (isolated AZPA4-MBP-6F6) was added to the left of the three fluorescent samples to demonstrate the difference in optical properties of isolated proteins. Right: 200 μl of 100mM imidazole elution fraction of MBP-YFP-AZPA4 was pipetted into Corning Costar 96-well white microtiter plate with transparent bottom to yield a pattern of "YFP". The plate was analyzed in DNA Bio-Imaging Systems box by illumination with the UV light.</td> 

   </tr>
 </tbody>

</table> <br> <br> <big><big><span style="color: black; font-weight: bold;">Characterization of MBP-RLuc-2C7<br> </span></big></big><span 

style="font-family: Arial;"><br>

<table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 400px; height: 307px;" alt=""
src="http://openwetware.org/images/2/21/MBPRLuc2C7biolum.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 26. MBP-RLuc-2C7 ZFP chimera exhibits bioluminsecence. </span>

40 μl of MBP-RLuc-2C7 supernatant (non-diluted, diluted 2 or 5-fold) was transferred to Corning Costar 96-well white microtiter plate. The bioluminescence assay was performed on a Berthold's ORION II Microplate Luminometer.</td> 

   </tr>
 </tbody>

</table> <br> <table style="text-align: left; width: 80%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
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     <td><span style="font-family: Arial;"><img
style="margin-right: 20px; width: 780px; height: 255px;" alt=""
src="http://openwetware.org/images/8/83/MBPRLuc2C7visual.png"></span></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-weight: bold;"></span><span
style="font-family: Arial;"><span
style="font-weight: bold;">Figure 27. Visual characterization of the activity of MBP-RLuc-2C7 chimera. </span>

Left: 100 μl of MBP-RLuc-2C7 supernatant was transferred to Perkin Elmer OptiPlateTM 96-well white plate in a pattern giving rise to the word "Luc". Right: 100 μl of the coelenterazine h substrate was added to the protein sample prepared in the same way as for the bioluminescence assay. Luminescence was detected in Syngene G:Box for 5 sec. <br><br></span></td> 

   </tr>
 </tbody>

</table> <br> <br> </span><big><big><span 

style="color: black; font-weight: bold;">BRET effect to detect neighboring binding sites on DNA target</span></big></big><span
style="font-family: Arial;"><br>

<br> <table style="text-align: left; width: 80%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr align="center">
     <td><span style="font-family: Arial;"><img
style="margin-right: 20px; width: 780px; height: 133px;" alt=""
src="http://openwetware.org/images/3/32/BRETDNAtarget.png"></span></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-weight: bold;"></span><span
style="font-family: Arial;"><span
style="font-weight: bold;">Figure 28: Sequence of the DNA target designed for BRET experiment. </span>

43 bp DNA target used to measure the BRET effect was designed as shown above. 2C7 binding sequence is colored blue and AZPA4 binding site in green. Spacer of 2 bp was selected to separate the binding sites and a clamp of the same length at both 5' and 3' end.<br><br></span></td> 

   </tr>
 </tbody>

</table> <br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 400px; height: 260px;" alt=""
src="http://openwetware.org/images/b/b8/BRETgraphDNA.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 29: BRET resulting from the addition of DNA target sequence to chimeric ZFP fusions.  </span>

mCitrine emission was normalized to the 535 nm emission of the MBP-RLuc-2C7. Since the emission spectra of RLuc and mCitrine overlap, 535 nm emission is observed for 100 nM MBP-RLuc-2C7 as well. Addition of 50 nM DNA target increased mCitrine emission indicating proximal binding of chimeric proteins to the DNA target.</td> 

   </tr>
 </tbody>

</table> <br> We demonstrated the design, production, purification and characterization of the two protein elements required for the reconstitution of the BRET sensor on a DNA origami. Initial experiments support the functional BRET, however the overlapping Renilla luciferase and mCitrine emission spectra suggest that selection of other BRET partners might improve the signal to background ratio, e.g. replace the Rluc-YFP pair with RLuc8-GFP2 BRET pair as a reasonable choice (De, 2007). Sensitivity of the BRET experiment on DNA origami could be improved by binding several neighboring BRET pairs on a single DNA origami rectangle.<br> <br> </span> <hr style="width: 100%; height: 2px;"> <ul> 

 <li><small>De A, Loening AM,

Gambhir SS. (2007) An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects. <span style="font-style: italic;">Cancer Research</span>, 67: 7175-7183.</small><!-- TU SE KONEA GLAVNO BESEDILO NA STRANI --></li> </ul> </div> </div> </div> <!-- end #content --> <div style="clear: both;">&nbsp;</div> </div> <!-- end #page --></div> <div id="footer-content" class="container"> <div id="footer-bg"> <table 

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