RCA Outputs Results

Linear DNA Probes vs Lettuce

Linear DNA Probes

Fluorophore (BBa_K4245130) and quencher-tagged linear DNA probes (BBa_K4245132) were used to quantify the presence and concentration of target microRNA (miRNA) in samples (see RCA: outputs). The decrease in fluorescence in the solution can be correlated with a specific concentration of miRNA through characterization. Similar to last year, we characterized and quantified rolling circle product (RCP) through the linear probes reporting mechanism (see Experiments: Linear DNA Probes with RCP). Resultant fluoresence was quantified in plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 1).

Figure 1. 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 (VarnBuhler et al., 2022). After the rolling circle amplification (RCA) reaction, the apatamer is able to bind to the RCP and produce fluorescence; therefore, an increase in miRNA concentration should correlate with an increase in fluorescence (see RCA: outputs). We characterized and quantified RCP through the Lettuce reporting mechanism (BBa_K4245134; BBa_K4245135) (see Experiments: Lettuce with RCP). Resultant fluoresence was quantified in plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 2).

Figure 2. 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.

RCA Optimization Results

Specificity

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.

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. 3) (see Experiments: blueGel™ with RCP).

Figure 3. Gel results: RCA with A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a; 2% agarose gel ran for 1 hour at 48V

We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader at an emission wavelength of 480 nm and an excitation wavelength of 528 nm (see Fig. 4) (see Experiments: Linear DNA Probes with RCP. 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 4. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes.

Emory Testing

In 2023, Lambert iGEM continued communication with Dr. Charles Searles of the Emory University in order to test whether our biosensor could be practical and applicable as a diagnostic tool. Researchers in the Searles Cardiovascular Lab ran RCA on 40.8 pM of miR-1 with SYBR™ Safe dye (see Experiments: SYBR™ Safe with RCP), which fluoresces when bound to ssDNA, as the output. As shown in Figure 5, there was a significant increase in fluorescence in the RCA reaction as compared to that of the controls, therefore validating the application of our biosensor in other labs.

Figure 5. Triplicate of RCA with SYBR™ Safe output done by independent hands

Ligation Time

Lambert iGEM’s 2022 RCA protocol (see Experiments: RCA 2022 protocol) requires samples to be incubated in the thermocycler at 37°C for two hours. However, the properties and usage of SplintR Ligase show that the reaction is successful with a 15-minute ligation time (Avantor Staff). Therefore, we ran RCA utilizing the miR-1 padlock probe (BBa_K4245200) with four different ligation times with miR-1 (BBa_K4245006): 15 minutes, 30 minutes, one hour, and two hours. After amplification, the reactions and controls were run on a gel; the bright bands near the top of the well showed that DNA product was produced for all reactions except for 15 minutes (see Fig. 6). Moving forward, we implemented a 30-minute ligation time (see Experiments: Optimized RCA protocol).

Figure 6. 1: 15-minute ligation, 2: 30-minute ligation, 3: 1-hour ligation, 4: 2-hour ligation, A: control (no enzymes); 2% agarose gel ran for 1 hour at 48V

Amplification Time

Lambert iGEM’s 2022 RCA protocol (see Experiments: RCA 2022 protocol) requires samples to be incubated in the thermocycler at 37°C for eight hours for amplification. To reduce this time, we ran RCA with two different concentrations of miR-1. After hybridization and ligation, we incubated the reactions in the plate reader at 37°C with 4uL SYBR™ Safe (see Experiments: SYBR™ Safe with RCP). The reactions were run overnight and the subsequent fluorescence was quantified in a plate reader (excitation wavelength 480 nm; emission wavelength: 528 nm): 30-minute intervals (see Fig. 7). Over time, the two RCA reactions increased in fluorescence, with no SEM overlap observed between the starting time and 5-hour mark. This suggests that RCP can be produced optimally starting at 5 hours. The significant increase in fluorescence between the RCA reactions and controls shows that SYBR™ Safe can determine the presence of RCP; however, the lack of difference between the 40.8 pM and .41 pM of miR-1 fluorescence indicates that the dye is not sensitive enough to differentiate between miRNA concentrations. Therefore, we did not continue to utilize SYBR™ Safe as a reporter.

Figure 7. RCA amplification optimization reaction with 40.8 pM and 0.41 pM of miRNA. No SEM overlap between starting point and 5-hour incubation time. SEM overlaps between 40. pM and .41 pM show that miRNA concentrations cannot be differentiated by SYBR™ Safe.

Phi29-XT

Lambert iGEM’s RCA protocol (see Experiments: Optimized RCA protocol) utilizes Phi29 DNA polymerase to perform amplification, resulting in an 5 hour amplification time. Phi29- XT DNA polymerase is an optimized enzyme with improved thermostability and sensitivity, which could shorten this time down to 2 hours (Biolabs). We ran RCA following the protocol for Phi29-XT on the New England Biolabs website (see Experiments: Amplification with Phi29- XT). The reactions and controls were run on a gel; no visible bands could be seen on the gel, indicating that RCP was not produced and therefore the reaction was not successful. As a result, we did not pursue utilizing Phi29-XT for further RCA reactions (see Fig. 8).

Figure 8. A: control (no enzymes), X: RCA with phi29-XT DNA polymerase; 2% agarose gel ran for 1 hour at 48V

Exponential RCA (eRCA) Results

Although the data from testing rolling circle amplification (RCA) with the target microRNA (miRNA) indicates high sensitivity — detecting lower limits of 2 pM — it lacks the ability to clearly differentiate between various concentrations of miRNA. Exponential RCA (eRCA) is an adaptation of RCA that produces exponentially greater fluorescence per unit of miRNA (Liu et al., 2013; Li et al., 2017); therefore, it has the potential to increase the distinction between lower miRNA concentrations and subsequently increase the margin of error (see RCA: eRCA).

RCA vs. eRCA

We performed eRCA with 40.8 pM of miR-1 (see Experiments: eRCA Protocol) then ran the reaction on a gel electrophoresis (see Fig. 9) (see Experiments: blueGel™ with RCP). Since eRCA produces multiple shorter strands of DNA (~25 nucleotides), we do not expect to see any bands on the 2% agarose gel. Therefore, the gel in Figure 9 indicates that eRCA was likely successful.

Figure 9. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see Experiments: eRCA Readout). The triplicate of eRCA with 40.8 pM of miR-1 significantly exhibits more fluorescence than that of the negative control (no enzyme), indicating that the eRCA reaction was successful (see Fig. 10).

Figure 10. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes); 2% agarose gel ran for 1 hour at 48V

In the future, we plan to test eRCA with the entire range of clinically relevant miRNA concentrations for coronary artery disease (CAD), as well as validate the applicability of eRCA with spiked serum samples. If the system proves to be more accurate than our current RCA biosensor, we will communicate with Dr. Charles Searles from the Emory University School of Medicine to test our biosensors in actual patient serum.

Capillary RCA Results

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) (see RCA: outputs).

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. 11) (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 11. 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. 12). 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 12. 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.

Inclusivity Estrogen RCA Results

We performed RCA on miR-20b, miR-328, miR-146a using our respective padlock probe designs (BBa_K4683008; BBa_K4683024; BBa_K4683028) and ran the subsequent rolling circle product (RCP) on a 1% agarose gel (see Fig. 2). The gel exhibited a fluorescent band of DNA very close to the well, indicating that a long strand of DNA greater than 1 kB - our RCP - was produced. Therefore, we can validate that our reaction was successful. In the future, we plan to expand our collection of biosensors to detect more miRNAs related to CAD and other demographics.

Figure 13. RCP from RCA reaction with miR-20b, miR-328,miR-146; 2% agarose gel ran for 1 hour at 48V

phi29 DNA Polymerase Protein Purification Results

After running our purified phi29 DNA polymerase from immobilized metal affinity chromatography (IMAC) on the Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)see Experiments: Protein Purification, Lambert iGEM saw bands around 50kD ~ 75kD. When we compared this band to the commercial phi29 DNA polymerase from New England Biolabs, it indicated that the protein was purified as the bands appeared in the same area (see Fig. 14).

Figure 14. SDS-PAGE with purified phi29 DNA polymerase, displaying bands at 50kD ~ 75kD

Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA) by comparing the rolling circle products (RCP) of the commercial and purified phi29 DNA polymerase. We added the same volume of both to RCA reactions and ran the rolling circle products on a gel. We saw fluorescent bands of DNA very close to the wells, indicating that both the RCA assay utilizing commercial and purified enzyme reactions were successful (see Fig. 15).

If we buy phi29 DNA polymerase from New England Biolabs, it will cost $251.00 for 1,250 units. However, after we utilized the NEBExpress Ni Spin Columns ($10 per prep to purify the enzyme), we were able to produce 200ul in one prep, or approximately 2,000 units. Because, it can be assumed that the unit between the commercially bought enzyme and our purified enzyme is similar, if commercially bought, this 200ul amount would cost around $400. Therefore, we can determine that our purified protein is significantly more affordable than the commercially bought protein.

Figure 15. Gel comparing the rolling circle product (RCP) between the team’s purified phi29 DNA polymerase and that commercially available phi29

In the future, we plan to more accurately quantify the activity of the purified units of phi29 DNA polymerase. Additionally, we hope to test the accuracy of our enzyme by quantifying the RCP using linear DNA probes (see RCA: outputs) to validate its use of point-of-care testing.

References