Biomod/2013/UT-Austin/Results

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Results

Linear Catalyst Controls

In order to ensure that the MDM2 and Ricin sequence adapted CHA circuit pieces function properly, H1, H2, and the reporter duplex were tested with a continuous catalyst oligo that contained all catalytic domains.

Figure 12: Fluorescence increases linearly with fluorophore concentration.
Figure 12: Fluorescence increases linearly with fluorophore concentration.


In order to relate fluorescence units with concentration of activated reporter, a standard curve (Figure 12) for the ricin generated apurinic site reporter fluorophore, BioM_RepFp1, was prepared. The linear regression for the above standard curve indicated that [BioM_RepFp1]=(Fluorescence Units-9.5238)/(12.019) with an R2 of 0.97. This equation was used to convert fluorescence values to concentrations for the continuous catalyst tests, Figure 13.

Figure 13: Increased concentration of the continuous catalyst, BioM_Cat_1,2,3p1, increases the rate of fluorescence development indicating increased concentrations of activated fluorophore and H1:H2.
Figure 13: Increased concentration of the continuous catalyst, BioM_Cat_1,2,3p1, increases the rate of fluorescence development indicating increased concentrations of activated fluorophore and H1:H2.


Fitting the data to a single exponential function, Figure 14A, allows us to assess the rate of signal amplification at various fluorophore concentrations, Figure 14B. Table 2 summarizes the data presented in Figure 14. The maximal observed rate, 0.1 min-1, is about a third of the lower observed rates from literature values, 0.35 min-1. The modified hairpin designs to adapt to the target sequence likely lowered the efficiency of signal amplification. These results still indicated that our ricin generated apurinic site hairpins function properly and should work with a split catalyst. Similar analysis was performed on the MDM2 SNP309 hairpins, and the results are summarized in Table 3.


Figure 14: The rate of fluorophore activation increases with increasing concentrations of catalyst. A: Curves are fit to the following equation: [Active Florophore]=aλt+c. B: The rate of fluorophore activation, λ (s-1), increases approximately linearly as catalyst concentration increases within this concentration range. However, the rate of increase decreases slightly within increasing catalyst concentrations as to be expected as the reaction approaches single turnover.
Figure 14: The rate of fluorophore activation increases with increasing concentrations of catalyst. A: Curves are fit to the following equation: [Active Florophore]=aλt+c. B: The rate of fluorophore activation, λ (s-1), increases approximately linearly as catalyst concentration increases within this concentration range. However, the rate of increase decreases slightly within increasing catalyst concentrations as to be expected as the reaction approaches single turnover.


Table 2:The values for Figure 14 are given below.
Rate vs Concentration for Apurinic Circuit
BioM_Cat_1,2,3p1 Concentration (nM) Observed Rate (min-1) Standard Error
0 0.0062 0.003
10 0.044 0.008
20 0.076 0.01
30 0.10 0.03


Table 3: The values for the kinetic analysis of the MDM3 circuit are given above. It can be seen that no signal is achieved above background. This indicates that the sequence adapted hairpin design is far too leaky to be a functional detection circuit.
Rate vs Concentration for MDM2 Circuit
BioM_Cat_1,2,3p1 Concentration (nM) Observed Rate (min-1) Standard Error
0 0.099 4.3
12.5 0.1 4.5
25 0.099 4.2

MDM2 Gene segment construction

In order to rapidly test MDM2 circuits on a sample MDM2 template, our team cloned a small 149 bp excerpt, Figure 15. This construction was performed through overlapping PCR, Figure 16. For long term storage and sequence confirmation, Figure 17, the purified full length template, was TA cloned into a pCR™2.1 backbone. Additionally, purified full length template was used to generate ssDNA for scCHA testing through assymetric PCR, Figure 18.

Figure 15: The constructed template is shown in the colored sequence above. The overlapping primer design for the template construction is shown below this sequence. The pink marker indicates the site of the T/G SNP.
Figure 15: The constructed template is shown in the colored sequence above. The overlapping primer design for the template construction is shown below this sequence. The pink marker indicates the site of the T/G SNP.
Figure 16: Overlapping PCR of the MDM2 gene excerpt oligos yielded products of mixed size. The 149 bp band was excised for cloning and sequencing.
Figure 16: Overlapping PCR of the MDM2 gene excerpt oligos yielded products of mixed size. The 149 bp band was excised for cloning and sequencing.
Figure 17: Sequencing chromatograms confirms that the MDM2 excerpt has been successfully constructed with the SNP alleles.
Figure 17: Sequencing chromatograms confirms that the MDM2 excerpt has been successfully constructed with the SNP alleles.
Figure 18: The ssDNA products of the assymetric PCR run faster than the dsDNA template.
Figure 18: The ssDNA products of the assymetric PCR run faster than the dsDNA template.


Conclusion and Future Direction

Apurinic site detection and allelic distinction remain the ultimate goal of this work. We have achieved a number of necessary steps to achieve this goal. We have determined that our hairpin design for the apurinic site detection circuit functions as expected with a continuous catalyst oligo. Further characterization of this circuit with the split catalyst and apurinic/purinic targets will need to be done. We have various MDM2 constructs built for testing, but the circuit design will need reworking.

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