Linear DNA Probes vs Lettuce

Lettuce DNA aptamer and linear DNA probes were both tested as reporters for rolling circle amplification (RCA) with the hsa-miR-1-3p padlock probe (BBa_K4245200); however, only linear probes were used for the successful characterization of miR-1 (BBa_K4245006) in the 2022 season (see Lambert iGEM Wiki RCA, 2022). Dr. Adam Silverman from Northwestern University and Dr. Mark Styzynski from the Georgia Institute of Technology both suggested utilizing an on-state reporter to make results more accurate and comprehensible; measuring gain of signal reduces fluorescent noise caused by the reaction and has less limitations compared to repressible systems. Linear DNA probes are an off-state reporter, therefore they produce an indirect relationship between microRNA (miRNA) concentration and fluorescence output. Utilizing an on-state reporter such as Lettuce would result in a direct relationship, in which a higher concentration of fluorescence will correspond to a higher concentration of miRNA.

Linear DNA Probes

Fluorophore and quencher-tagged linear DNA probes were used to quantify the presence and concentration of target miRNA in samples. Each probe contains part of the complement to the middle sequence (BBa_K4245131) of the rolling circle product (RCP): One is tagged with the fluorophore dye FAM (BBa_K4245130), and the other is tagged with the quencher molecule BHQ1 (BBa_K4245132). The fluorescent signal from the fluorophore is shut off by the quencher as a result of a fluorescence resonance energy transfer (FRET) reaction (Zhou et al., 2015). The reaction is distance-dependent; when the quencher and fluorophore-tagged linear probes bind to the RCP, they are in close proximity (see Fig. 1), allowing non-radiative energy to be transferred from the excited fluorophore to the quencher (Sekar & Periasamy, 2003). The decrease in fluorescence in the solution can be correlated with a specific concentration of miRNA through characterization.

Figure 1. Linear DNA probes binding to RCP, leading to diminished fluorescence

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. 2). Changes in fluorescence were measured using a plate reader in experimentation: the emission spectrum of FAM is 480 nm in wavelength, while the excitation spectrum is 528 nm in wavelength (Zhou et al., 2015).

Figure 2. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes

Lettuce

Lettuce is a fluorescent DNA aptamer that binds with the dye DFHBI-1T within its secondary structure, thus causing the dye to fluoresce (see Fig. 3) (VarnBuhler et al., 2022). The split Lettuce design includes two halves of the Lettuce aptamer and their flanking sequences. After the RCA reaction, we add the split Lettuce sequences and the DFHBI-1T dye to the RCP. The left flanking sequence (BBa_K4245134) will bind to the first half of the middle sequence of the RCP, and the right flanking sequence (BBa_K4245135) will bind to the second half of the middle sequence. Once together, the dye is able to bind and produce fluorescence, therefore an increase in miRNA concentration should correlate with an increase in fluorescence.

Figure 3. Split Lettuce aptamer with DFHBI-1T binding to RCP

We characterized and quantified RCP through the Lettuce reporting mechanism (see Experiments: Lettuce with RCP). There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 4). Changes in fluorescence were measured using a plate reader in experimentation: the emission spectrum of DFHBI-1T is 480 nm in wavelength, while the excitation spectrum is 528 nm in wavelength (VarnBuhler et al., 2022).

Figure 4. Characterization curve showing a positive logarithmic relationship between miR-1 concentrations and RFU from split Lettuce aptamer

Comparison

Both reporter mechanisms resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. However, there was no indication that Lettuce outperformed linear DNA probes. Therefore, we continued to conduct further experiments with linear probes. 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.

Capillary

To create a more accessible miRNA detection system, Lambert iGEM also adopted another output approach: capillary-rolling circle amplification (cpRCA). This method offers faster reaction times and eliminates the need for expensive and specialized equipment like plate readers and fluorometers, making it suitable for rapid point-of-care testing of miRNAs (Hixson & Ward, 2021). Unlike our current reporter mechanisms, cpRCA amplifies miRNA to produce RCP within a glass capillary tube. The small diameter of the capillary tube (0.1mm) enables diffusion of small polymers (such as miRNA), while restricting diffusion of larger polymers (such as the RCP). Due to this principle (see Modeling), the miRNA molecules are relatively spaced apart within the tube (see Fig. 5a). As the RCP is being synthesized from the miRNA molecules, the large DNA strands are unable to diffuse, creating spaced apart, isolated regions of ssDNA (see Fig. 5b). When the DNA fluorescent dye SYBR™ Safe is present in the reaction solution, it binds to the synthesized RCP to create regions of high fluorescence, or fluorescent “dots” (see Fig. 5c), a phenomenon that has been explored with PCR in capillary tubes (Choi et al., 2018). We can directly quantify the number of miRNA molecules by counting the number of dots, which can be done through use of a phone camera and counting algorithm, such as Open Computer Vision Library (OpenCV) (Abid et al, 2021).

Figure 5. (a) miRNA and padlock probe diffusion in capillary tube. (b) Isolated regions of RCP within the capillary tube. (c) SYBR™ Safe fluorescence with RCP

After discussion with Dr. Charles Searles from the Emory University School of Medicine, we determined that the upper limit of clinically relevant miRNA concentrations in patients with CAD is around 40 pM. We initially ran cpRCA with the miR-1 padlock probe (BBa_K4245200) and 40.8 pM of miR-1 (BBa_K4245006) but were not able to visualize any dots of miRNA within the capillary tube (see Fig. 6) (see Experiments: Capillary RCA). This is likely because the higher concentration of miRNA resulted in a significant overlap of fluorescent regions within the capillary tube, leading to inaccurate quantification through cpRCA.

Figure 6. Visualization of cpRCA with 40.8 pM of miRNA

To ensure the amplified products were countable, we diluted the sample down to 1.66 fM, or approximately 50 molecules of miRNA. To validate that cpRCA would still be successful at this lower concentration, we ran a gel electrophoresis on the RCP from the cpRCA reaction after 4 hours of amplification (see Experiments: blueGel™ with RCP) using both 40pM and 1.66fM of miRNA to observe the difference. By analyzing the results on the gel, we concluded that RCP was likely produced with both concentrations as the gel exhibited fluorescent bands of DNA very close to the wells, while no band was expressed in the control (see Fig. 7). As a result, we can infer that cpRCA was successful after 4 hours of amplification and at only 50 molecules of miRNA, which is more efficient than the 8 hour amplification of traditional RCA, and increases the sensitivity of the assay.

Figure 7. Lane 1: ladder, Lanes 2-3: cpRCA with 1.66 fM miR-1, Lanes 4-5: cpRCA with 40.8 pM miR-1, Lanes 6-7: control; 2% agarose gel ran for 1 hour at 48V

In the future, we plan to conduct further experimentation with lower miRNA concentrations to better visualize the fluorescent regions in the capillary tube, as well as characterize the full range of relevant miRNA concentrations for CAD.