RCA
Overview
Problem
Heart disease has been one of the most prevalent diseases in the United States, with 1 in 4 deaths caused by cardiovascular disease (Centers for Disease Control, 2022). According to the Centers for Disease Control and Prevention, heart disease has contributed to over 25% of all deaths in Georgia, Lambert iGEM’s home region, in 2018 - that is 5% greater than the overwhelmingly large national statistic. Historically, the southern region has had issues concerning obesity, high blood pressure, smoking, and diabetes, all of which are exacerbated by a family history of coronary artery disease (CAD). These problems only increase with the “coincidental” ethnic and racial minorities present within these low-income populations (Hames and Greenlund). However, with a process that considers both affordability and accuracy, we can proliferate the means of diagnosing CAD. This starts with inclusive initiatives targeting these overlooked demographics.
Methodology
Last year, Lambert iGEM created CADlock, an accessible point-of-care diagnostic tool to both detect and quantify microRNA (miRNA) by utilizing rolling circle amplification (RCA). The rolling circle product (RCP) is a continuous transcription of the team’s designed padlock probe, which consists of many interspaced repeats of the reporting mechanism and target miRNA’s complementary sequence (Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016). We identified two miRNAs found to be upregulated to CAD, hsa-miR-1-3p (BBa_K4245006) and hsa-miR-133a-3p (BBa_K4245009), to serve as an early indication of the disease (Kaur et al., 2020).
Initially, we considered Reverse Transcription Quantification PCR (RT-qPCR) which was appealing due to its regularity with current screening, especially with the COVID-19 tests. However, through conversations with experts and further analysis of protocols, we identified the dangers of this method due to the high rate of false positive mismatches and determined that RCA held the most advantages. Not only does RCA eliminate the use of primers–which RT-qPCR requires–but it has no data concerning any mismatch or inconsistent results from our literature studies (Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016).
Moreover, diagnostic tools such as echocardiograms and angiograms are expensive – costing upwards of $1000 – and not readily available at most primary care facilities, preventing a large portion of the population from receiving effective screening (Hansing C. E.). However, our RCA assay costs approximately $9, which is less than 1% of most common diagnostic procedures.
Padlock Design
A padlock probe is a single-stranded DNA sequence (30-150 nucleotides in length) designed to hybridize to a specific RNA/DNA target– in this case, our target miRNA (Jonstrup et al., 2006). The ends or “arms” of the probe are complementary to the target sequence, which allows the miRNA to bind antiparallel to the padlock arms (see Fig. 1). We designed these ends to have similar annealing temperatures to maximize their binding efficiency with the miRNA. In between the ends is the reporter sequence, which can be utilized to quantify the initial miRNA concentration (see Fig. 1). Furthermore, we added a phosphate group modification to the 5’ end of the padlock sequence (Jonstrup et al., 2006).
The following steps are for generating a padlock probe by hand. Users will need a software tool such as SnapGene that displays melting temperatures of sequences.
- Paste target sequence in software of choice.
- Take the reverse complementary sequence of the target.
- Split the sequence in half. Use the first half of the sequence as the end of the padlock probe and the second half as the beginning of the probe.
- Insert the desired reporter sequence in between the two halves of the sequence.
- Calculate the annealing temperature of both halves and move nucleotides one at a time from one end of a half to the end of the other half until the difference between the annealing temperatures of the two arms is minimal.
Last year, Lambert iGEM developed Probebuilder: a novel, intuitive, and user-friendly program for designing padlock probes. After validating the hsa-miR-451a padlock probe generated by Probebuilder, the team has continued to use the software to design padlocks for RCA reactions (see Lambert iGEM Wiki Software, 2022).
Probebuilder’s source code is available on the 2022 Gitlab repository
Process
The RCA process relies on the continuous replication of padlock probes to produce RCP that can be quantified and correlated to the target miRNA concentration. There are three main steps in the RCA process detailed by Jonstrup et al. in their 2006 paper: hybridization, ligation, and amplification. By designing the padlock probe to detect a specific miRNA target, the miRNA and padlock probe arms form a DNA-RNA hybridization (see Fig. 2a, 2b). The arms are then in closer proximity, and the enzyme SplintR ligase (which only ligates near RNA-DNA hybridization) utilizes a phosphate modification on the 5’ arm and ATP to circularize the padlock probe (Avantor Staff) (see Fig. 2c). After ligation, phi29 DNA polymerase uses the miRNA hybridized to the padlock arms as a primer to initiate and perform amplification (see Fig. 2d, 2e). The resultant rolling circle product (RCP) contains interspaced repeats of the middle sequence of the padlock probe (see Fig. 2f). This sequence of the RCP is complementary to the reporters (linear probes or lettuce) used to correlate fluorescence to varying concentrations of the target miRNA.
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2022 Results
In 2022, we conducted multiple rounds of experimentation to generate quantifiable results with our product. First, we conducted initial experiments to assess how well our hsa-miR-1-3p biosensor (BBa_K4245200) performs in a controlled laboratory setting by evaluating the effectiveness of rolling circle amplification (RCA) and various reporter mechanisms. All results in Figures 4-11 and13 were quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 518 nm.
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To quantify the rolling circle product (RCP), we first used the fluorescence dye SYBR™ Safe dye which fluoresces when bound to ssDNA. The results were inconsistent, so we tested the potential rolling circle product (RCP) through gel electrophoresis. The gel showed a band near the wells, indicating a long strand of DNA was produced, likely our RCP (see Fig. 3).
Figure 3. Gel electrophoresis of RCA reactions, depicting bands greater than 1 kB; 2% agarose gel ran for 1 hour at 48V -
Through a literature review, we found a lettuce aptamer (BBa_K4245133) and tested it with simulated RCP (BBa_K4245131). This showed an increase in fluorescence compared to the controls (see Fig. 4), indicating that the lettuce aptamer is able to fluoresce when bound to the simulated RCP (VarnBuhler et al., 2022).
Figure 4. Lettuce aptamer with simulated RCP. -
We performed a series of experiments of the split Lettuce aptamer (BBa_K4245134; BBa_K4245135) with actual RCP. The results display a significant increase in fluorescence with RCP and split lettuce as compared to the controls, indicating that the lettuce aptamer was able to successfully bind to the RCP and induce fluorescence of DFHBI-1T (see Fig. 5).
Figure 5. Graph of fluorescence from left to right: 1) water + dye control, 2) split lettuce + dye control, 3) split lettuce + RCP + dye, 4) split lettuce + padlock&miRNA control + dye. -
After consultation with other experts on linear DNA probes, we decided to test these with simulated RCP (BBa_K4245131) to ensure linear probes were an effective and characterizable means of quantifying miRNA (Zhou et al., 2015). The FAM dye tagged probe (BBa_K4245130) and BHQ-1 quencher tagged probe (BBa_K4245132)) are complementary to our RCP. When the linear DNA probes are bound to the RCP, the fluorescence produced by the FAM Dye is quenched by the BHQ-1 quencher which significantly reduces fluorescent intensity (Zhou et al., 2015). As shown in Figure 6, linear DNA probes with simulated RCP have significantly less fluorescence than just the FAM dye, indicating the mechanism would be efficient with actual RCP.
Figure 6. Linear DNA probes with simulated RCP. -
We further characterized the linear DNA probes with various simulated RCP concentrations. Figure 7 below shows a negative logarithmic correlation between the complement concentrations and the relative fluorescence units, a parallel relationship to our ODE model (see Fig. 8).
Figure 7. Linear DNA probes with various concentrations of simulated RCP.
Figure 8. Deterministic ODE Model Simulation of RFU output dependent on concentration of linear DNA probe complement concentration. -
After we determined the vitality of using linear DNA probes with simulated RCP, we tested these probes with actual RCP. As shown in Figure 9 below, there is a statistically significant decrease in the fluorescence output of the probes with RCP as compared to that of just the FAM-tagged probe. This confirms that we produced our desired RCP.
- We further characterized linear DNA probes to quantify the relationship between various miRNA concentrations and fluorescence. There is a negative logarithmic correlation between the miRNA concentrations and the RFU (see Fig. 10), a parallel relationship to our ODE model (see Fig. 11), validating that RCA coupled with linear probes are an effective means of quantifying miRNA.
Serum Testing
To assess the feasibility of our biosensors in real-world applications, Lambert iGEM experimented with pooled human serum. Upon meeting with Dr. Charles Searles from the Emory University School of Medicine, we tested the ability of our RCA sensors to detect miRNAs in blood serum by spiking pooled human serum with hsa-miR-1-3p and RNase inhibitors (to prevent native RNases from degrading the added miRNA). The gel electrophoresis results show clear bands close to the wells (see Fig. 12), indicating our biosensors were able to produce a long strand, likely the RCP.
To experimentally validate that the long DNA strand that was produced is our desired RCP, we utilized linear DNA probes. As shown in Figure 13, there is a statistically significant decrease in the fluorescent output of the linear DNA probes with RCP as compared to that of just FAM tagged probes, validating the success of the RCA reaction with hsa-miR-1-3p in spiked serum.
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.
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).
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.
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).
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).
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.
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.
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.
Specificity
While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, 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. 2) (see Experiments: blueGel™ with RCP).
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. 3) (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.
Emory Testing
Dr. Charles Searles is a cardiologist specialist working with biomarkers regarding coronary artery disease (CAD) and an associate professor of medicine at Emory Healthcare Hospital. Last year, he provided us with hsa-miR-451a (BBa_K4245012) - which is unrelated to the pathophysiology of CAD - as a control for our proof of concept experimentation and also offered to test our biosensor in his lab. This year, we continued to communicate with him in order to test whether our biosensor could be practical and applicable as a diagnostic tool. This was done with the help of Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, who ran the RCA reactions utilizing our protocol and miR-1 padlock probe (BBa_K4245200), and their enzymes (see Fig. 4) (see Experiments: SYBR™ Safe with RCP). They ran RCA on 40.8 pM of miR-1 (BBa_K4245006) with SYBR™ Safe dye, which fluoresces when bound to ssDNA, as the output. Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. As shown in Figure 4, 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.
After running our assay twice, Ms. Rooney gave us feedback regarding our protocol such as reducing the number of variables as controls and performing serial dilutions for more precise concentrations of miRNA. Ultimately, she told us that our biosensor lacked practicality and applicability for point-of-care testing due to its long reaction time, which is approximately 15-16 hours‒ more than double the time needed for quantification through qRT-PCR. Subsequently, the next step we took to optimize RCA was to reduce the time required to run the reaction.
Time Optimization
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. 5). Moving forward, we implemented a 30-minute ligation time (see Experiments: Optimized RCA protocol).
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. 6). 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.
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. 7).
Overall, we were able to reduce the RCA workflow from around 15 hours to 7 (see Experiments: Optimized RCA Protocol).
Exponential Rolling Circle Amplification (eRCA)
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. Throughout multiple iterations of testing, Lambert iGEM has consistently found a significant overlap in error bars for the fluorescent output, especially among lower concentrations of miRNA. Despite taking extra steps to decrease potential error (see RCA: Optimization), we still see a large margin of error. After further discussions with Dr. Mark Styczinski from the Georgia Institute of Technology, we concluded that this overlap in error bars is likely caused by the similarity of fluorescent outputs ‒ due to the minute scale of and minimal difference between concentrations ‒ rather than experimental variation. 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 decrease the margin of error.
RCA vs. eRCA
There are two major differences between eRCA and RCA: the padlock probe design and the amplification process. The RCA padlock probe consists of two parts: the end or “arms” of the padlock probe and the middle sequence (see RCA: 2022 Overview). The arms are the reverse complement of the target miRNA, while the middle sequence corresponds to the reporter mechanism (Jonstrup et al., 2006). The three main steps of RCA are hybridization, ligation, and amplification (Jonstrup et al., 2006) (see RCA: 2022 Overview). The target miRNA first hybridizes to the padlock probe, bringing the two ends of the probe together. Then, the two ends are ligated by SplintR ligase. The bound miRNA serves as a primer for Phi29 DNA polymerase, initiating the amplification of the padlock probe. The result is a long repeating strand of DNA, known as the rolling circle product (RCP), used to quantify the initial concentration of the miRNA (Jonstrup et al., 2006). In eRCA, the padlock probes consist of an additional part between the arms and the reporter sequence: the complement of the endonuclease binding site (see Fig. 1) (Li et al., 2017). When the RCP is synthesized during amplification, the nicking endonuclease Nb.BbvCI will recognize this site and cleave the outer strand of DNA, or the RCP, while it is still bound to the padlock probe (Biolabs). This ensures that the padlock probe remains intact while releasing individual strands of the synthesized miRNA and reporter sequence. The produced miRNA can then bind to the padlock probe and initiate the eRCA process again, essentially creating an endless loop of amplification until the reagents are consumed or the reaction is deactivated (see Fig. 2) (Liu et al., 2013; Li et al., 2017). The exponential increase in output can increase the differences in fluorescence emission between miRNA concentrations, subsequently decreasing the margin of error and accuracy of our overall biosensor.
Lettuce is a fluorescent DNA aptamer that binds with and induces the fluorescences of the DFHBI-1T dye (VarnBuhler et al., 2022). Initially, we considered producing whole Lettuce aptamers through the RCA reaction. However, Dr. Mark Styczsinski and Megan McSweeney from the Georgia Institute of Technology advised us against this as the secondary structures of the aptamers would likely interfere with each other on a single DNA strand. As a result, we chose to pursue other reporter mechanisms with the traditional RCA assay (see RCA: Outputs). Contrary to RCA, eRCA produces multiple isolated strands of the reporter sequence, offering the potential to create whole Lettuce aptamers without the risk of secondary structure interference (Liu et al., 2013; Li et al., 2017). The hsa-miR-1-3p eRCA padlock probe (BBa_K4683002) consists of the following parts: 3’ arm for miR-1 (BBa_K4245100), 5’ arm for miR-1 (BBa_K4245107), Lettuce aptamer complement (BBa_K4683000), and Nb.BbvCI binding sites (BBa_M31961). As the concentration of the target miRNA increases, the padlock probe produces more miRNAs and Lettuce aptamers, resulting in an exponential increase in fluorescence output in the presence of DFHBI-1T.
Results
We performed eRCA with 40.8 pM of miR-1 (see Experiments: eRCA Protocol) then ran the reaction on a gel electrophoresis (see Fig. 3) (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 gel. Therefore, the gel in Figure 3 indicates that eRCA was likely successful.
We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see Experiments: eRCA Readout). Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. The triplicate of eRCA with 40.8 pM of miR-1 exhibits significantly more fluorescence than that of the negative control (no enzyme), indicating that the eRCA reaction was successful (see Fig. 4).
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.
Overview
One of Lambert iGEM’s major goals in 2023 was to increase the inclusivity of our biosensors for all demographics. Through literature review, we found that healthcare settings tend to emphasize responsive care for men, resulting in lower patient retention among women, especially during coronary artery disease (CAD) diagnosis (Johns Hopkins, 2018). We validated this disparity with Dr. Mindy B. Gentry, a cardiologist in Georgia, and Dr. DeLisa Fairweather, a director of research at the Department of Cardiovascular Diseases at Mayo Clinic. To further address this issue, we specifically targeted microRNAs (miRNAs) correlated to CAD in women (see Inclusivity: Estrogen). Discussion with these experts confirmed the use of hsa-miR-20b (BBa_K4683005) as a target for our rolling circle amplification (RCA) assay. Moreover, we extended the correlation of miRNAs to CAD through specific racial groups, utilizing CADmir to identify hsa-miR-328-3p (BBa_K4683021) and hsa-miR-146a (BBa_K4683025). Further collaboration with Dr. Charles Searles, a professor of medicine at Emory Healthcare Hospital, enabled us to utilize hsa-miR-150-5p (BBa_K4683009), hsa-miR-122-5p (BBa_K4683013), and hsa-miR-30c-5p (BBa_K4683017) as they correlate with CAD in African American populations. Therefore, we can incorporate these miRNAs into our research to further accommodate an inclusive approach for understanding and detecting CAD.
Estrogen miRNA
Women after menopause are more likely to develop heart disease due to a decrease in estrogen hormone levels (Brandt, 2013). Studies conducted by Pare et al. in 2022 found that estrogen directly suppresses CAD through the Estrogen Receptor α (ERα) pathway; when estrogen is present in the bloodstream, it activates the ERα pathway, subsequently activating the production of CAD suppressors. Post-menopausal women have lower levels of estrogen within their bloodstream, resulting in the inhibition of CAD suppressors and therefore increasing patient susceptibility to CAD (Brandt, 2013; Pare et al., 2022). The concentration of hsa-miR-20b is positively correlated with that of estrogen (Pérez-Cremades et al., 2018). Like the estrogen molecule, miR-20b activates the ERα pathway, regulating the production of CAD suppressors (Pérez-Cremades et al., 2018). Dr. Fairweather validated the use of this miRNA to detect the risk of CAD in post-menopausal women (see Inclusivity: Estrogen).
Other miRNAs
We conducted a comprehensive literature review using CADmir to efficiently identify relevant miRNAs (see Fig. 1) (see Software: CADmir). In just 4 minutes and 12 seconds, we uncovered three miRNAs and a selection of related articles.
One of these miRNAs, hsa-mir-328-3p, was found to be correlated with cardiovascular disease, specifically atherosclerosis. Therefore, it serves as a valuable predictive tool for early detection of heart disease, with a specific focus on the African American population (Iwańczyk, 2023). Similarly, hsa-mir-146a is a miRNA associated with the regulation of inflammation and CAD, predominantly linked to the Latin American population (Iwańczyk, 2023). These findings are of particular relevance to our team after considering the demographic makeup of Georgia. Notably, the African American and Latin American population have experienced significant growth, as demonstrated by the statistic: “the Black (non-Hispanic) population had the most growth increasing by 518,670 from 2.9 million in 2010 to 3.5 million in 2021” (Georgia Population by Year, County, Race, & More, 2023).
To further complement our findings, we consulted with Dr. Searles, who recommended three additional miRNAs correlated with CAD in African American populations: hsa-mir-150-5p, hsa-mir-122-5p, and hsa-mir-30c-5p.
Gel Experimentation
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.
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