Biomod/2011/TUM/TNT/Results
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TUM NanU - Home
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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>|
<a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Fluorescence_Measurements">Fluorescence Measurements</a>|
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Results
Survey of Results
We successfully designed a structure that folds properly with high yields (link) and is suitable for observing structural deformations. Comprehensive TEM analysis yielded insights into global structural deformations and allowed for statistical evaluation of angle and length distributions dependent on DNA binder concentrations (link). Our structure was successfully labeled with fluorescent dyes and a considerable variety of different approaches to fluorescence measurements was tested. In single molecule measurements FRET events could be observed. (link). Based on these experimental data as well as our structure simulations and calculations, we gained new insights into the structural properties of DNA origamis especially with regards to binding of small molecules (link).
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 properly 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, not better, results as the 15_65 ramp, indicating an efficient folding even after such short time. |  |
| Most of the structures from the major band were folded correctly, which was demonstrated by TEM imaging (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 definitely higher, since even weak dilutions led to appropriate concentrations for TEM and fluorescence microscopy. Therefore we estimate the yield to be roughly 10 nM. |  |
TEM Image Analysis
Distribution of Angles
When we inspected the structure in the TEM, we saw a spread of the arms in the upright projections. (figure 3). The extent of this spread seemed to be correlated to the amount of DNA binding molecules.
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DNA binders were added in such concentrations to theU, that a previously calculated fraction of binding sites should be occupied (see here for calculation). 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 spermine, ethidium bromide and DAPI as well as the negative control and the positive control (intrinsically twisted due to additional base pairs) are displayed in table 1.
<html> <center><font size=1><b>Table 1: Results from twist measurements</b></font></center> <table cellspacing="0" cellpadding="6" align="center"> <tr bgcolor="#f5f5f5"> <td></td><td><b>Number of particles</b></td><td><b>Mean angle [degree]</b></td></td><td><b>Variance</b></td></td></td> </tr> <tr bgcolor="#f5f5f5"> <td>negative control</td><td>715</td><td>9.0</td><td>4.1</td>> </tr> <tr bgcolor="#f5f5f5"> <td>positive control (pretwisted)</td><td>424</td><td>21.1</td><td>5.8</td> </tr> <tr bgcolor="#f5f5f5"> <td>Spermine 0.42 µM (in average one molecule per 21bp)</td><td>1019</td><td>8.7</td><td>3.8</td> </tr> <tr bgcolor="#f5f5f5"> <td>Spermine 1.34 µM (in average one molecule per 7bp)</td><td>1809</td><td>9.3</td> <td>4.5</td> </tr> <tr bgcolor="#f5f5f5"> <td>Spermine 6.7 µM (in average one molecule per 2bp)</td><td>621</td><td>12.5</td> <td>4.5</td> </tr> <tr bgcolor="#f5f5f5"> <td>Spermine 12.1 µM (in average one molecule per 1.5bp)</td><td>1129</td><td>12.3</td> <td>2.5</td> </tr> <tr bgcolor="#f5f5f5"> <td>Ethidium bromide 0.74 µM (in average one molecule per 21bp)</td><td>626</td><td>11.2</td> <td>3.3</td> </tr> <tr bgcolor="#f5f5f5"> <td>Ethidium bromide 2.27 µM (in average one molecule per 7bp)</td><td>652</td><td>8.6</td> <td>5.4</td> </tr> <tr bgcolor="#f5f5f5"> <td>DAPI 144 nM (in average one molecule per 21bp)</td><td>325</td><td>7.4</td> <td>3.2</td> </tr> <tr bgcolor="#f5f5f5"> <td>DAPI 432 nM (in average one molecule per 7bp)</td><td>375</td><td>6.0</td> <td>3.8</td> </tr> </table> </center> </html>
The values in table 1 are based on the following histograms (figure 4):
The distribution of angles in the negative control has two populations, one where the two arms are exactly above each other which leads to very small angles and one where the two arms are modestly spread. This leads to a gaussian distribution around a finite angle. The width of this distribution is in good agreement with the calculated thermal fluctuations, which yield deviations of approximately 4.4°.
| The positive control with an internally induced twist due to 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) displays much larger angles (see figure 5). The population around zero is probably caused by deformed structures which had no second arm but could not be visually excluded. This results in many angles around zero. The other population around the finite angle is now the more spread structure. Here the angle of the positive control is shifted to larger 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 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 for 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 basepair in the base of theU.
For small spermine concentrations, no significant peak shift can be observed. Only at higher concentration, the maximum of the distribution is shifted noticeably towards higher angles. For the series negative control - ethidium bromide every 21 bp - ethidium bromide every 7 bp, the data display no systematic shift, while for DAPI, the mean angle decreases continuously.
Length Measurements
We also measured the lengths of the origami structures on TEM images. Histograms of the length distributions display a gaussian shape (figure 6). For increasing concentrations of spermine, the length decreases steadily (figure 6a, for raw data see this file: <html><a href="http://openwetware.org/images/b/b6/TEM_length_measurements_raw_data.xlsx">TEM_length_measurements_raw_data.xlsx</a></html>. Surprisingly, for rising concentrations of ethidium bromide, the length decreases as well, although addition of ethidium bromide is usually known to increase the length of a simple double stranded DNA (figure 6b). It seems that DNA origami structures behave different from single DNA helices in this regard.
 |  |
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 not successful. The photospectrometer turned out not to be 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 7, 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.
 |  |
To handle the issue of small concentrations, further experiments were performed with a fluorescence microscope.
Single Molecule Measurements at the Fluorescence Microscope
FRET Measurement
A typical FRET-trace can be seen in the following video which also plots the donor, acceptor and FRET intenities over the time.
<html><center><iframe width="425" height="349" src="http://www.youtube.com/embed/wyt8qzZpRog?hl=de&fs=1" frameborder="0" allowfullscreen></iframe></center></html>
| The analysis program is a matlab script 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 (see figure 9). The graph shows the intensities of the donor and the acceptor and, in addition, the intensity of the FRET-events. As one can see, the intensity of the donor rises as soon as the acceptor bleaches. After some while the donor bleaches too. From that, the FRET-efficiency can be calculated. First we measured the FRET-efficiencies for the BM14 structure without any intercalator or groove binder as a control and afterward we measured the same structure with 4.8µM spermine (corresponding to one molecule every 7bp). The FRET-efficiencies were plotted in figure 10. |  |
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It is obvious that we actually measured FRET, though the low yield of FRET-events that were found by the matlab script does not allow to draw any conclusions because of low-number statistics. The wide spread of FRET efficiencies is probably caused by the base twists observed in the TEM measurements. Here further optimization is neccessary. Yet the fact that there actually were FRET-events makes it worth to keep on elaborating these measurements.
Fluorescence Tracking
Apart from FRET-measurements, we also applied another approach to investigate the deformation of the structure where we determined the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first, we excited the Atto 550 dye and observed at its characteristic wavelength, subseqeuntly Atto 647N was excited and observed. For the analysis with the homemade matlab script, at first we had to calibrate the cameras.
Then the matlab script searched for spots in the green and the red picture and fitted them with a gaussian. The peaks from the green picture are subsequently overlaid with the red picture. When there is a matching red spot for the green spot the distance between them is calculated.
We did those measurements for a control and for two different concentrations of spermine.
Quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm. Nevertheless, we decided to take pictures in epifluorescence mode of a negative control without DNA binders and with two different spermine concentrations (one spermine every 7 bases and one spermine every 21 bases). Every picture was illuminated for 1 sec with the green laser for the green channel and then with the red laser for the red channel for the same time. The graph below (figure 11) shows the histograms of the distribution of the distance between the maxima of the fitted gaussians in the green and red channel.
The distributions look nearly the same for every concentration, except for the control. This is due to the small number of points that were measured for these traces. Furthermore, the values for each trace seam not to be distributed in a gaussian manner. This maybe underlies the electrostatic repulsion of the arms when they are in close vicinity. Also the distribution reaches up to 120 nm. This is not realistic. Possible reasons for this artifacts could be misalignments of the pictures and not accurate enough determination of the spots since we wanted to measure spatial separations in the regime of 5 nm which corresponds to a 20th of one single pixel on the detector. Also acquisition of uncorrelated spots which belong to different structures might be a problem. So one has to refine the setup and acquire more values for better statistics to get trustable values of a mean distance of the arms.
Discussion
Origamis Respond in Another Way than Single DNA Helices on Local Deformations
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.). 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.
Although both DNA binders induce length changes in opposite directions on DNA helices, both shorten the whole origami structure. The crosslinking between the helices in theU alters the type of deformation compared to an isolated double helix. One could assume that local changes in twist and length combine in an origami, causing a length change effect with all local deformations integrated.
Regarding the measured twist angles, for small concentrations no effects can be seen with spermine. Without spermine, as well with ca. 5% and 14% occupied binding sides, the angle remains ca. 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 helix 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.
To put these considerations in a nutshell, new theoretical approaches are needed to correlate effects on a single helix with effects on a huge system of interconnected helices.
Twisted Positive Control is good Comparison for Deformation by Ethidium Bromide
One approach to gain further insights and a solid experimental fundament for this goal was the investigation of an intrinsically twisted structure as positive control. In average every 21bp an additional base was inserted, resulting in global deformations that were easily observable in the TEM. Effects on length cannot be examined in this way, since the positive control needed a longer scaffold than the normal theU structure, but it is a good examination object for the angles between the arms. We compared the data with those from ethidium bromide, since every bound ethidium bromide as well as every additional base cause comparable elongation and they differ only in the twist they cause on a double stranded DNA. Thus this effect can be examined isolated. 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. One could argue that our method is error-prone due to the angle measurement by hand, but the width of the distributions is in good agreement with the calculated thermal fluctuations, so these data can be regarded as reliable. It will be necessary to check further DNA binders, but the direction of twist should be of high importance for the angle deformation. Positive twists add to the existing pitch, while the negative twist by ethidium bromide needs to work against the intrinsic direction of helical rotation. One needs to consider also that the direction of the total twist of the structure cannot be determined from the 2D projections analyzed in this study. Therefore, FRET measurements would be an appropriate method.
New Practical Methods and Theories will be needed
Although we cannot present final results for FRET analyses, first single molecule analyses can be provided. For an optimization of the FRET studies, the origami structure needs some slight improvements, like a more rigid base or fluorophores attached nearer to the base. For this optimization, we have laid a thorough fundament not only of experimental results, but also lots of theoretical considerations, which can explain flexibility and correlate observable (via TEM and / or fluorescence measurements: distances, angles) with unobservable (twists) structural changes.
On the experimental side, one could try to eliminate some uncertainties regarding the applied concentrations. We did some calculations to determine the fraction of occupied binding sites even at small concentrations, but as mentioned above, binding could be cooperative and for a proper testing of such a behavior, concentrations of bound DNA binders must be checked experimentally. This is very trying due to the small concentrations and the little fraction of compounds bound compared to those free in solution. We suggest to try some radiolabeled DNA binders, of which the bound fraction can be determined from radioassays.
Outlook
By exploiting the potential of our device to gather new knowledge about DNA-small molecule interactions, it should be possible to unravel structural deformations on even tinier levels. This information does not only allow a more sophisticated understanding of the flexibility of origamis in response to varying triggers, but also helps elucidating the mechanic and maybe also mechanistic effects of DNA binders on DNA.
For this we envisioned a characteristic plot for DNA binders based on the twist and length changes they cause, as depicted in the sketch below (figure 12). Following the example of the famous Ramachandran plot, which enables bioinformaticians to predict secondary structure motifs of proteins with high accuracy, a plot of twist vs. length changes could designate certain regions of increased occurrence. In these regions, the effects of binding molecules within a common binding class would gather. With a modified structure where fluorophore positions have been optimized according to a refined theory of deformation and with an appropriate knowledge base of structural changes due to well-characterized binders, an easy and probably high-throughput procedure for the screening of potential DNA binding molecules could be in closer reach. The folding of already well-designed DNA origamis is, in contrast to the design itself, rather straightforward and requires only basic equipment. By customizing design, folding and purification processes, a wider application would be possible and, as such, attractive e.g. for basic research or pharmaceutical drug development.
Another intriguing feature of our findings is that, with the proper refinements to the underlying model, it should be possible to create a device whose conformational changes can be precisely predetermined. As a result, this would permit to use the principle the other way around. Knowing the outcome of conformational changes of DNA origami using a certain concentration of a well known DNA-binder will provide a valuable tool, advancing the development of custom-made dynamic structures from DNA origami. By altering the concentration of an appropriate binder, movements of susceptible origami parts could be triggered, with the option to reverse to the original state through withdrawal of the binder.
