Part:BBa_K4245130:Experience

Linear Probes with Complement

Lambert_GA 2022
We initially tested linear probes with the complement of the middle sequence to ensure that linear probes were an effective and characterizable means of quantifying miRNA (see RCA Protocols)

Figure 1. Linear Probe Complement Fluorescent Readout

Figure 1 displays a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and Linear Probe Complement as compared to a triplicate of just FAM tagged Probes. Therefore, we concluded that linear probes were an efficient means of reporting the output of our biosensor.

Figure 2. Characterization curve for parts BBa_K4245130 and BBa_K4245132 showing a negative logarithmic relationship between RFU and complement concentrations ranging from 0.1-100 μM. Note: 0-0.1 μM shows a positive relationship, but large error bars at 0 μM suggest this was due to faulty pipetting.

Figure 3. Deterministic ODE Model Simulation of RFU output dependent on the concentration of linear DNA probe complement concentration.

In order to quantify the relationship between linear probe complement concentration and fluorescence, we further characterized these parts with varying linear probe complement concentrations. There is a negative logarithmic correlation between the complement concentrations ranging from 0.1-100 mM and the relative fluorescence units (RFU) (see Fig. 2). The 0 mM complement concentration outputs less RFU than 0.1 mM, which does not align with the model. However, the large error bars at 0 mM suggests that there was some degree of significant error. Thus, this data point is insignificant and further trials should be performed to achieve more accurate results. Moreover, the data from 0.1-100 mM closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 3) correlating complement concentration to RFU (see Model). Therefore, the overall data collected depicts an accurate relationship between the complement concentration and RFU.

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 linear probes are efficient means to do the aforementioned tasks through testing with the complement of the linear probes, we wanted to confirm that they are able to quantify the miRNAs experimentally (see RCA Protocols)

Figure 4. Fluorescent Read of Rolling Circle Product for miRNA-133-3p and miRNA-1-3p

As shown by Figure 4, 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 for our miRNA-1-3p and miRNA-133a-3p sensors and that this mechanism was an effective reporting method for our sensor.

Figure 5. Fluorescent reading of RCP produced by miR-451a padlock probe (generated via Probebuilder). BHQ-1 and FAM probes compared to solely FAM probes.

Figure 5 displays a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and the RCP produced as compared to a triplicate of just FAM tagged Probes. This finding experimentally validates the use of ProbeBuilder as a means of producing effective padlock probes.

Figure 6. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and miRNA concentrations

Figure 7. Deterministic ODE Model Simulation of RFU output dependent on concentration of miRNA concentration.

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. 6). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 7) correlating complement concentration to RFU (see Model). 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 8. 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.

In addition, Lambert iGEM also tested linear probes in spiked human serum (where we added miRNA-1 to serum) in order simulate human serum. As shown by Figure 8, 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.

Lambert_GA 2023
Linear DNA Probes

Linear DNA probes were tested as reporters for rolling circle amplification (RCA) and was used for the successful characterization of hsa-miR-1-3p in the 2022 season (see Lambert iGEM Wiki RCA, 2022). Linear DNA probes are an off-state reporter, therefore they produce an indirect relationship between microRNA (miRNA) concentration and fluorescence output.

Similar to last year, we characterized and quantified RCP through the linear probes reporting mechanism (see Experiments: Linear DNA Probes with RCP). There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 9).

Figure 9. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes

Conclusion Because other reporter mechanisms did not outperform linear probes, we continued to conduct further experiments with them. In the future, we hope to find another on-state reporter that would make reading RCA results more comprehensible and accurate. Such reporters include molecular beacons and other DNA aptamers.

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