Part:BBa_K4245107:Experience

Applications of BBa_K4245107

Lambert_GA 2022 This part was experimented with as a part of the composite parts, hsa-miR-1-3p RCA Padlock Probe (BBa_K4245200) and hsa-miR-1-3p RCT Padlock Probe (BBa_K4245201). RCA with BBa_K4245200 Rolling Circle Amplification (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

Therefore, RCA created RCP that can be quantified by our chosen reporting mechanisms.
RCT with BBa_K4245201 Rolling Circle Transcription (RCT) was run with this part but proved to be unsuccessful. The products of RCT are long RNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCT can be determined is by running the RCT products on an agarose gel, since a fluorescent band very close to the wells would indicate the presence of extremely long nucleic acid strands. However, the products of RCT did not appear on a 1% agarose gel (see Fig. 1).

Figure 1. Picture of RCT products (circled in green) run on a 1% agarose gel. There were no visible bands that indicate the production of long RNA strands.

The RCT products were also tested with DFHBI-1T, a fluorophore that fluoresces when reacting with the Broccoli RNA fluorescent aptamer. DFHBI-1T was added to the RCT products, and the fluorescence was read on the plate reader.

Figure 2. Graph of fluorescence output before and after the addition of DFHBI-1T to RCT products and controls.

However, when we added DFHBI-1T to the RCT products, there was no significant increase in fluorescence observed as compared to the controls (see Fig 2.).

From these results, Lambert iGEM determined that the process of RCT itself was not successful in our lab. We also tested the folding of the aptamer with the spacer with our BBa_K4245210 experimentation. Linear Probes with RCP
We use linear probes as a means to quantify and report the miRNAs that we sensed through rolling circle amplification (RCA) reactions. To go beyond verifying that are efficient means to do the aforementioned tasks through testing with complement of the linear probes, we wanted to confirm that they are able to quantify the miRNAs experimentally (see RCA Protocols)

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

As shown by Figure 1, 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 for our miRNA-1-3p and miRNA-133a-3p sensors and that this mechanism was an effective reporting method for our sensor.

Figure 2. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

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. 12). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 13) correlating complement concentration to RFU (see Modeling).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.

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