Biomod/2011/TUM/TNT/Results: Difference between revisions
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<h3>Fluorescence Tracking at the Fluorescence Microscope</h3> | <h3>Fluorescence Tracking at the Fluorescence Microscope</h3> | ||
Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we directly | Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength. | ||
For the analysis with the homemade matlab script at first we had to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/10/19#Camera_calibration calibrate the cameras]. | For the analysis with the homemade matlab script at first we had to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/10/19#Camera_calibration calibrate the cameras]. | ||
Then the matlab script searches for spots in the green and the red picture and fits in an gaussian. The peaks from the green picture then are transfered into the red picture. When there is a matching red spot for the a green spot the distance between them is calculated. | Then the matlab script searches for spots in the green and the red picture and fits in an gaussian. The peaks from the green picture then are transfered into the red picture. When there is a matching red spot for the a green spot the distance between them is calculated. | ||
We did those measurements for a control and for two different concentrations of spermin. | We did those measurements for a control and for two different concentrations of spermin. | ||
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<a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification">Folding & Purification</a>|
<a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#TEM_Image_Analysis">TEM Image Analysis</a>|
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Results
http://openwetware.org/index.php?title=Biomod/2011/TUM/TNT/Results&action=edit
Folding & Purification
The U structure was folded using the 15_65 ramp. This ramp was the fastest of the tested ones and also led to proper folded origamis as shown in figure 1. There is only one major band visible in the agarose gel, indicating that no significant amounts of byproducts (like dimers) have been formed. The results of the slower ramps 2D_H3_ML and 5D_H3_ML yielded similar results as the 15_65 ramp.
Most of the structures from the major band were folded correctly, which was proven by TEM images (figure 2).
The purification of the structures was tried both with an agarose gel and with an Amicon size exclusion filter (molecular weight cutoff: 100kDa). According to general experience, the yield of purification via agarose gel is approximately 2 nM. The yield of the filter purification was definitively higher, since unexpected weak dilutions led to appropriate concentrations for TEM and fluorescence microscope. Therefore we estimate the yield to be roughly 10 nM.
TEM Image Analysis
When we inspected the structure in the TEM, we saw a spread of the arms in the uprightly orientated structures (figure 1). The magnitude of this spread seemed to be correlated to the amount of DNA binding molecules in solution.
The angles between the arms were measured with different concentrations of DNA binders. The widths of the angle distributions could be explained by thermal fluctuations. We assumed to find a peak shift of the angles dependend on the added DNA binder concentration.
The peaks for the tested DNA binding molecules DAPI, ethidium bromide and spermine as well as the negative control and the positive control (intrinsically twisted) are displayed in table 1.
<html> <center><font size=1>'''Table 1: '''</font> <table cellspacing="0" cellpadding="6" align="center"> <tr bgcolor="#f5f5f5" > <td><b></b></td><td><b>Number</b></td><td><b>Mean</b></td></td><td><b>Variance</b></td></td><td><b>SEM</b></td></td> </tr> <tr bgcolor="#f5f5f5"> <td>ctrl</td><td>715</td><td>9.312</td><td>4.830</td><td>0.180642978</td> </tr> <tr bgcolor="#f5f5f5"> <td>ctrl pretwisted</td><td>424</td><td>21.119</td><td>8.161</td><td>0.396347765</td> </tr> <tr bgcolor="#f5f5f5"> <td>Spermine 0.42 µM</td><td>1019</td><td>8.864</td><td>4.793</td><td>0.150157669</td> </tr> <tr bgcolor="#f5f5f5"> <td>Spermine 1.34 µM</td><td>1809</td><td>9.663</td> <td>5.759</td><td>0.1354099</td> </tr> <tr bgcolor="#f5f5f5"> <td>EtBr 0.69 µM</td><td>156</td><td>9.169</td> <td>5.036</td><td>0.403186678</td> </tr> <tr bgcolor="#f5f5f5"> <td>EtBr 0.74 µM</td><td>472</td><td>11.059</td> <td>5.577</td><td>0.256697629</td> </tr> <tr bgcolor="#f5f5f5"> <td>EtBr 2.27 µM</td><td>223</td><td>10.877</td> <td>5.531</td><td>0.370363066</td> </tr> <tr bgcolor="#f5f5f5"> <td>EtBr 2.4 µM</td><td>431</td><td>7.474</td> <td>7.839</td><td>0.377567259</td> </tr> <tr bgcolor="#f5f5f5"> <td>DAPI 432 nM</td><td>375</td><td>6.0277</td> <td>5.3736</td><td>0.277491511</td> </tr> <tr bgcolor="#f5f5f5"> <td>DAPI 144 nM</td><td>325</td><td>7.5568</td> <td>4.1349</td><td>0.229362984</td> </tr> </table> </center> </html> !!!Include ausagekräfitge Bilder von gespreizten U's, eine reihe control, andere twisted control!!! Fig. 1: The class averages of pure structures (left) and a structure with an internal twist, induced by additional base pairs (right). A spread of the arms in the twisted structure is clearly visible.
Histogramms
The values in the above mentioned table 1 come from the following histograms:
Distribution of Angles
The measured angles are distributed in a gaussian manner around an angle [math]\displaystyle{ \phi_0 }[/math] with a width [math]\displaystyle{ \sigma }[/math]. The distribution of angles in the control has two populations. One were the two arms are exactly above each other which leads to very small angles and one were the slightly twisted arms are pulled down to the surface at the adheration to the grid and therefore is pushed to the side and repelled from the lower arm. This leads to the distribution around an finite angle as one can see in fig. 2. Further more we measured a structure with an internally induced twist by including additional base pairs in each helix (these additional base pairs lead to a net torque in each helix and therefore a macroscopic deformation of the structure) which lead to a distribution of the angles around a much higher angle (see fig. 3). The population around zero is maybe due to deformed structures which had no second arm and couldn't be excluded. This results in many angles around zero. The other population around the finite angle is now the more spread structure. This angle is shifted to higher values by approximately a factor of 2 because of the induced twist. So in principle this way of measuring the deformation of our structure in dependence of induced stress works.
The width of our measured angles can be explained by the following mechanism:
when the grids for TEM are prepared, the structures are able to fluctuate around a certain mean position which - in our case - corresponds to [math]\displaystyle{ \phi_0 }[/math]. So when the structures adhere to the carbon film of the grid and stain is added, they are fixed in one actual position. Since this fluctuation can be described by a Boltzmann distribution, we can easily calculate a theoretical value for the width of our angle measurements with some assumptions (for more details, please see: Thermal fluctuation of the arms).
So we get a theoretical prediction of
[math]\displaystyle{ \sigma = 3.9^{\circ} }[/math]
which approximately explains the width of our measurements which can be seen in fig. 2 and 3.
File:Control with dblgauss.png
Fig. 2: Histogram of the structure without any twist or DNA binding molecule. The two populations were fitted with a sum of two gaussians with a width around [math]\displaystyle{ \phi_1 = ... }[/math] of [math]\displaystyle{ \sigma_1 = ... }[/math] and another width of [math]\displaystyle{ \sigma_2 = ... }[/math] around [math]\displaystyle{ \phi_2 = ... }[/math]. The total number of measured structures was n=... .
File:Control twist with gauss.png
Fig. 2: Histogram of the structure an internal induced twist. The population around the finite angle was fitted with a gaussian with a width around [math]\displaystyle{ \phi = ... }[/math] of [math]\displaystyle{ \sigma = ... }[/math]. The total number of measured structures was n=... . A shift to higher angles is clearly visible.
.png|x400px]]
Fig. 4:
We then plotted a histogram of the mean angles versus the part of the structure occupied with DNA binders. We could clearly see a rise of the spread with rising concentration of DNA binding molecules in fig. 5. Since we had many particles, the error is relatively small compared to the values and we assumed our mean spread angle to be accurate.
Length measurements
We also measured the lengths of the origami structures on the TEM images. Histograms of the length distributions display a gaussian shape (figure xxx). For increasing concentrations of spermine, the length decreases steadily (figure yyy). Surprisingly, for rising concentrations of ethidium bromide, the length decreases as well, although addition of ethidium bromide is known to increase the length of a simple double stranded DNA. It seems, that origami structures respond in another manner then single helices.
figure xxx: image length distribution histogram with gaussian fit, negative control
figure yyy: concentration series length gaussian fits, left: spermine, right: ethidium bromide
TIRF?
Including Base-Twist Theory
The measured angles φ for negative and positive control, [math]\displaystyle{ \phi_{neg} \approx 9 }[/math]° and [math]\displaystyle{ \phi_{pos} \approx 21 }[/math]°, can be related to a torsion α of the base according to the Theoretical considerations of the base twist:
[math]\displaystyle{ \frac{cos \alpha -1}{\alpha} = \frac{B L}{R (B - 2 L)} sin \frac{\phi}{2} }[/math]
The theory determines the torsion for these particular φ-values to [math]\displaystyle{ \alpha_{neg} \approx 33 }[/math]° and [math]\displaystyle{ \alpha_{pos} \approx 93 }[/math]°. This corresponds to a torsion of 5° per base-pair in the base.
Fluorescence Measurements
FRET Bulk Measurements
For first tests, a simple 18 bp DNA double helix with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined. The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was unsuccessful. The photospectrometer is not sensitive enough to handle Atto dyes at concentrations below 10 nM (peaks were not visible at all). The real time PCR, which is more sensitive, still did not deliver trustworthy data when using 50 µl samples with 10 nM Atto dyes. It could be shown that the reproducibility of the real time PCR setup was poor with deviations of up to 40 % between identical samples (figure 1) . To assure the identity of the samples a 100 µl stock was divided into two 50 µl samples. Based on these results no experiments with theU structure were performed at all with this device as the concentration of theU structure is lower than the concentration of the here test structure.
Figure 1: FRET efficiency Spermine and FRET efficiency EtBr
To handle the issue with the small concentrations further experiments were done with a fluorescence microscope.
FRET at the Fluorescence Microscope
FRET Measurement
We designed the structure in such a way that a small change of angle in the base, which is a 30 helix bundle in a honey comb lattice, is amplified by the two arms, which are both 10 helix bundles and therefore should twist as well. To measure the change in twist and angle two fluorophores were attached to the two arms so that a deformation should cause a change in distance between them. We chose a donor and an acceptor fluorophore, namely Atto 550 and Atto 647N, so a change in distance between them leads to a change in FRET-efficiency.
In order to immobilize our structure standing upright on the coverslide we used neutravidin and biotinylated oligos complementary to staples at the base of our structure, which is a common way to immobilize DNA origamis on surfaces.
To prepare the slides we used this procedure.
The fluorescence microscope has three lasers with different wavelenghts (blue:473nm, green: 532nm, red: 640nm). We only used the red and the green one because of the dyes we attached to our “U”.
For the measurement we used alternating-laser excitation of single molecules (ALEX) with an excitation length of 0.05 sec.
... film einfügen?!? ...
Depending on the background we decided to use the microscope either in epifluorescence or in TIRF modus.
The analysis program is a matlab program which searches for spots in the red and the green movie and plots the intensities over time to identify bleaching events. Only those plots where the acceptor bleaches first and the donor bleaches afterwards are useful to calculate the FRET-efficiency.
Fig: Example of an intensity over time plot of the acceptor and donor: !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
The analysis of all movies is summarized in the following histogramms
.... Histogramme ...
Fluorescence Tracking at the Fluorescence Microscope
Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength. For the analysis with the homemade matlab script at first we had to calibrate the cameras. Then the matlab script searches for spots in the green and the red picture and fits in an gaussian. The peaks from the green picture then are transfered into the red picture. When there is a matching red spot for the a green spot the distance between them is calculated. We did those measurements for a control and for two different concentrations of spermin.
Though quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm a qualitative evidence can be see in a shift in distance from the control to higher concentrations of spermin of approximately nm. This shows that in principle it is possible to detect a structure deformation of our biosensor.
Discussion
Spermine causes a positive twist (46°) of double stranded DNA, and additionally decreases the length of DNA (base step rise reduced from 0.34nm to 0.29nm; Tari et.al.). Length measurements based on our TEM images also indicate a decrease of the total length of the origami structure. Said changes are in good agreement. One spermine every 7bp reduces the length of a single helix to 97.9% of the original length, while the origami is shortened to ca. 98.6% by the same occupation with spermine. The changes are small, but local length changes seem to add up to comparable global changes.
Regarding the measured twist angles, for small concentrations no effects can seen with spermine. Without spermine, as well with ca. 5% and 14% occupied binding sides, the angle remains around 9°. For higher occupations (50% and 67%), the angle increases to 12°. Additional data points will be needed to fit these findings, but we suggest that a cooperative behavior would be an appropriate explanation. Within DNA origamis, not only a single helices needs to be twisted, but large bundles of helices with many crosslinks. This makes the single helices more rigid, consequently hindering an induced fit of spermine molecules. Only higher concentrations could excert enough force to overcome the local restraints and induce a global twist.
According to Salerno et.al., each bound molecule of ethidium bromide increases the length of a DNA double helix by 3.4nm, which is exactly the length of one base pair. Additionally, it induces a twist of -27°, in contrast to the +36° twist of one base pair. Regarding our angle distributions from the TEM data, the mean global twist for one additional base every 21bp is 21°, compared to 11° induced by one molecule ethidium bromide every 21bp. Besides some potential inaccuracies in the quite small concentrations, the strong local negative twist caused by ethidium bromide results in remarkably reduced effects on the global structure.
Outlook
Referring to the relevance of this project accordingly the future goal should be to find a way to characterize DNA binding molecules. In the field of bioscience the Ramachandran plot is a nice way to show typical secondary structures of proteins.
In this case we would plot twist against length (fig 1). To do this it would be necessary to test more DNA binding molecules and to design a structure which is more sensitive to measure changes in length. This should be done based on the theoretical background which considers geometrical thoughts and information we gained during this project.
By optimizing the structure based on our results it should be possible to create a device whose conformational changes can be precisely predetermined. This would permit to use this structure the other way around. Knowing the outcome of conformational changes of DNA origami using a certain concentration of an well known DNA-binder will provide a valuable tool especial in the field of creating functional DNA origami.
Welche Experimente wären noch möglich/ sollten noch gemacht werden, um... zu zeigen. Ferne und nahe Zukunft. Was könnte damit noch untersucht werden (Sequenzspezifische Bindeproteine...)
- Struktur mit asymmetrischem feature um die richtung der projektion bestimmen zu können
