Part:BBa_M31961:Experience

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Applications of BBa_M31961

Lambert_GA 2023
A padlock probe, which is often 30-150 nucleotides in length, is a single-stranded DNA sequence designed to recognize a specific target sequence. The “arms” of a padlock probe are the ends of the ssDNA that are complementary to a specific target sequence. The middle sequence (the sequence between the arms) can be specifically designed to perform a function once amplified. (Nilsson et al., 1994)
Lambert iGEM found hsa-miR-1-3p (BBa_K4245006) to be upregulated in correlation to CAD (Kaur et al., 2020). For miRNA 1, we designed two complementary arms, BBa_K4245100, the 3' arm for hsa-miR-1-3p and BBa_K4245107, the 5' arm for hsa-miR-1-3p, with Nb.BbvCI nicking sites BBa_M31961 before the 3' arm and after the 5' arm. For the reporter, we decided on the complement of the Lettuce Aptamer BBa_K4683000 so that during the amplification process, Lettuce Aptamers would be produced (see fig. 1).

Figure 1. eRCA padlock design
<br< References
Kaur, A., Mackin, S. T., Schlosser, K., Wong, F. L., Elharram, M., Delles, C., Stewart, D. J., Dayan, N., Landry, T., & Pilote, L. (2019). Systematic review of microrna biomarkers in acute coronary syndrome and stable coronary artery disease. Cardiovascular Research, 116(6), 1113–1124. https://doi.org/10.1093/cvr/cvz302

Nilsson, M., Malmgren, H., Samiotaki, M., Kwiatkowski, M., Chowdhary, B. P., & Landegren, U. (1994). Padlock probes: Circularizing oligonucleotides for localized DNA detection. Science, 265(5181), 2085–2088. https://doi.org/10.1126/science.7522346

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. 2).

Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)
Figure 2. 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. 3).

Figure 2. Graph of fluorescence after DFHBI-1T was added.
Figure 3. 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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