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Experimental data (provided by the 2013 Stanford-Brown iGEM Team)

Thermal denaturation: thermodynamic evidence of silver binding

For this experiment, we sought to unequivocally prove that there was silver uptake by the excised duplex. To do this, we annealed the strands in two conditions: Ag+ (silver present) and Ag- (silver absent). The mismatch-complementary oligonucleotides in this sample were shown to only anneal in the presence of Ag+.

It is known that when double-stranded DNA is heated, it “melts,” or experiences a breakdown of its secondary structure. Beer’s law states that for a fixed volume and molecule, absorbance is proportional to concentration. As DNA melts, the concentration of nucleotides doubles, and the absorbance will increase similarly.

To show intercalation, we hypothesized that in this highly-mismatched strand, annealing will only occur in the presence of silver. Consequently, those strands should only show an absorbance increase with temperature if they were annealed with silver. That which does not anneal will not melt.

The graph below is the average of four trials, plotted for unit-less change in absorbance at 260nm (absorbance of dsDNA) over increasing temperature. The spectrophotometer we had access to restricted temperature to a maximum of 45°C, which we factored into the sequence design. 535 data points for each trial were taken across a 20°C temperature difference, rendering the ensuing data quite statistically significant.

Figure 1: average of four trials on 32bp10CC Star sequence, P value of 10^-272 in paired T-Test.

PAGE: electrical analysis of duplex formation

We utilized poly-acrylamide gel electrophoresis (PAGE)to produce visual representations of duplex formation, and we used this method to demonstrate that duplexing occurs as a 1:1 molar ratio between CC mismatches and silver ions is reached.

We tested undersaturation (<1:1 Ag:CC) and oversaturation (>1:1 Ag:CC) of the system, and showed the duplexing point. Duplexing became visually apparent at the 1.5:1 silver to mismatch ratio, which is slightly higher than expected. Nonetheless confirms both the role of silver in annealing mismatch-complementary strands and that the set point of the system is near a 1:1 ratio.

Figure 2: 10% polyacrylamide gel in MOPS buffer, 32bp10CC strand with increasing molarity of silver (inset above lanes); duplexing begins to be visible at 1.5:1 silver to mismatch ratio-- while this is not ideal, it demonstrates visually the duplexing as it occurs; duplexes are the bands above the rest, indicative of the size difference that comes with duplexing.

TEM: Visual analysis of particle pairing

We used transmission electron microscopy (TEM) to visualize our DNA directly. We hoped to directly identify the presence of silver in our samples. This was easier said than done, yet we used software to analyze the images that suggests that such a feat is possible.

We used a sputtering technique to coat our samples with platinum ions. This allowed for a better visualization of the DNA, though it provided no analytical data (platinum masks the silver). We were excited by the striated nature of the DNA, again confirming that we were able to form duplexes. This DNA was supercoiled due to the sample preparation procedure, but was intriguing nonetheless.

We then used a negative staining technique to visualize different samples to extract information about the structure of the strands. The high resolution images showed a mass of DNA, but we were able to analyze these images using ImageJ, an NIH-provided software for image analysis.

Under TEM, electron-dense areas such as silver are expected to shine brightly, while the low-electron DNA is expected to show up as a dark spot. BBa_K1218026 has a silver ion pattern of 2-1-2:

5’ AAA CAT TTA CCA CCT CCT CCA CCT TAT AGT TT 3’
   ||| ||| ||| ••| ••| ••| ••| ••| ||| ||| ||
3’ TTG TTA AAT CCT CCA CCA CCT CCA ATA TCA AA 5’

Thus, where silver is present inside the DNA, it should be at a known distance from other particles. With this in mind, we used ImageJ to find all particles within 30% of the silver ion size (2.4Å diameter, or 5.8Å2). We then measured the distance of the center of each particle from nearby particles. As expected, we saw local maxima at the distances representing distances of one and two base pairs (3.4Å and 6.8Å, respectively). We did this measurement manually using the software, and are in the process of using Matlab code to do the same process, albeit at a higher fidelity. Local maxima at integer multiples greater than two base pairs were not observed, likely due to the supercoiling of the DNA.

In sum, by using TEM, we were able to visually demonstrate the silver ion distribution across our samples, providing even further evidence of the robustness of our system.

Figure 3(a): Platinum sputtering TEM image of 32bp10CC strand (BBa_K1218026); of note is the striated pattern in the DNA; Figure 3(b): negative staining technique of different sample of 32bp10CC: dark areas are DNA, light areas are electron-dense areas; Figure 3(c): image threshold of 3(b) taken using ImageJ; Figure 3(d): particles delineated as ranging between 0.45 and 0.75 Å^2; Figure 3(e): paired particle counting of 3(d) using ImageJ and manual measurements, though rudimentary, the trend was strong: 124/647 particles were within 2.5-4.5 angstroms of one another, corresponding to roughly the length of one nucleotide.

Figure 3(f) Using a similar method but a tighter particle size (0.45-0.65 Å^2), distances between the centers of particles were counted and placed on a histogram; note local maxima at 3.4 and 6.8 angstroms, 1 and 2 bases in length, respectively.

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