Part:BBa_K4245100:Experience

Applications of BBa_K4245100

Lambert_GA 2022 Rolling Circle Transcription (RCA) was successful with this part. The products of RCA are long DNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCA can be determined is by running the rolling circle products (RCP) on an agarose gel. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, our RCP was run on a gel. The result was a really long DNA strand close to the well.

Figure 1. Image of gel ran with miRNA-1 RCP product.

By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced. The gel exhibited a fluorescent band of DNA very close to the well, which indicates that a long strand of DNA, greater than 1 kB, was produced due to our reaction (see Fig. 1). As a result, we can infer that the RCA reaction allowed the creation of a really long DNA stand -- our RCP.
The RCP was also tested with the split lettuce aptamer. DFHBI-1T and the lettuce right and the modified lettuce left was added to the RCP, and the fluorescence was read on the plate reader.

Figure 2. Graph of split Lettuce reaction with RCP. The values represent the change in fluorescence before and after the reaction with DFHBI-1T took place.

As seen in Figure 2, the increase in fluorescence of the RCP + Lettuce + dye was significantly greater than the controls, which suggests that the split Lettuce was successfully bound to the RCP. In addition, the DFHBI-1T was also successfully bound within the split lettuce secondary folding. According to these results, RCA was successful, and the reaction between the split lettuce and RCP was successful as well.

In addition, to split lettuce, RCA and RCT were also tested with the FAM and BHQ1 labeled linear probes.

As shown by Figure 3, there is a statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction and that this mechanism was an effective reporting method for our sensor.

Figure 3. Fluorescent Read of Rolling Circle Product for miRNA-133-3p and miRNA-1-3p

In order to quantify the relationship between miRNA concentration and fluorescence, we further characterized these parts with varying linear probe complement concentrations. There is a negative logarithmic correlation between the complement concentrations and the relative fluorescence units (RFU) (see Fig. 4). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 5) correlating complement concentration to RFU. Therefore, the overall data collected depicts an accurate relationship between the miRNA concentration and RFU, further validating that RCA coupled with linear probes are an effective and efficient means of quantifying miRNA concentrations.

Figure 4. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

Figure 5. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

In addition, Lambert iGEM also tested linear probes in spiked human serum to simulate human blood. As shown by Figure 6, there is statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction performed on our miRNA-1-3p spiked serum. This further validates that biosensors utilizing RCA coupled with FAM and BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.

Figure 6. Linear DNA Probe Fluorescence from Serum Extracted miRNA-1-3p Rolling Circle Amplification. Results show significant decrease in fluorescence, indicating a successful Proof of Concept.

Therefore, RCA created RCP that can be quantified by our chosen reporting mechanisms.


Lambert_GA 2023
Specificity Testing
While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock BBa_K4245200 in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence ,BBa_K4245006, which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV, BBa_K4683003, and one with three SNVs, BBa_K4683004. hsa-miR-133a-3p, BBa_K4245009, was also included to ensure the padlock would not ligate to any miRNA.

Figure 1. Comparison of sequences used to test specificity of hsa-miR-1-3p RCA padlock: 1 bp SNV in the seed region, 3 SNVs outside of seed, and miR-133a-3p

We ran the reactions and control on a gel electrophoresis; only the well with 40.8 pM of miR-1 showed visible bands of DNA near the top of the wells, which is likely our RCP (see Fig. 2). We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader (see Fig. 3) The RCA reaction utilizing the miR-1 padlock probe with miR-1 exhibited significantly less fluorescence than the other miRNAs. Since linear DNA probes produce a negative correlation between fluorescence and miRNA concentration, this result, along with the gel, indicates that RCA is specific to single nucleotide differences.
Figure 2. Gel results: A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a- Image of an agarose gel run with RCP from RCA run with 40.8 pM of each miRNA in reaction.
Figure 3. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes

eRCA
Exponential Rolling Circle Amplification (eRCA) was successful with this part. The products of eRCA are short DNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCA can be determined is by running the exponential rolling circle products (eRCP) on an agarose gel. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, no/dim bands near the top of the well indicate that short DNA was produced(see fig. 1).

Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

By analyzing the results on the gel, our team concluded that short strands of DNA were produced, likely the eRCP.
The eRCP was also tested with DFHBI-1T dye as the RCP would consist of Lettuce Aptamer sequences. The fluorescence was read on the plate reader (see fig. 2).

Figure 2. Graph of fluorescence after DFHBI-1T was added

As seen in Figure 2, the increase in fluorescence of the eRCP+dye was significantly greater than the controls, which suggests that Lettuce aptamers were produced. According to these results, eRCA was successful.

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