RCT

Overview

Our project for the 2022 iGEM competition provides a point of care (POC) diagnostic tool for coronary artery disease (CAD) using circulating miRNA in blood as biomarkers.

The Problem

In the United States, 1 in 5 deaths are due to cardiovascular disease (Centers for Disease Control, 2022). One of the most common heart diseases is coronary artery disease (CAD), accounting for 17.8 million annual deaths worldwide (World Health Organization, 2022). CAD is especially prevalent in the Southeastern United States, our iGEM team’s home region. In order to address this issue, we have developed a point-of-care (POC) diagnostic tool to detect and quantify microRNA (miRNA) by utilizing rolling circle amplification/transcription from a rolling circle product. This product is created by continuous transcription of the designed padlock probe to create long repeats containing complementary sequences to the miRNAs and the reporting mechanism. The reporting mechanisms emit fluorescence from the repeating product, which can then be quantified by a frugal device, MicroQ. In addition to other existing screening methods, this technique can aid healthcare professionals in the diagnosis of disease as results rely on the interpretation and laboratory work best suited to healthcare facilities. Moreover, this year’s team hoped to alleviate the high costs associated with current early diagnostic measures and provide an additional screening tool for physicians.

Methodology

Lambert iGEM identified two specific miRNAs — miRNA 1 and miRNA 133a— found to be upregulated in the correlation to CAD (Kaur et al., 2020). The proposed method aims to detect a certain threshold of these miRNAs to provide an early indication of CAD. We considered several methods to detect and quantify miRNA including toehold switches, RT-qPCR, padlock probes and SHERLOCK systems, summarized in Figure 1. However, consultations with experts in their field such as Dr. Koob (an expert in SHERLOCK) and further analysis on the protocols for each methodology revealed that the use of padlock probes would be best suited in detecting miRNAs. The short sequence of each miRNA and repeating nucleotides made them difficult to quantify and interfered with hybridization. Ultimately, we chose Rolling Circle Amplification (RCA) and Rolling Circle Transcription (RCT) due to the ability of padlock probes to detect the short sequences of miRNA and discriminate between different miRNAs with single-nucleotide selectivity. Through RCA/RCT and the respective reporting mechanisms, the miRNAs present in blood serum can eventually be detected and quantified.

Other Methods

Lambert iGEM chose the rolling circle methods instead of other methods due to their precision and easily quantifiable results (see Fig. 1). These include:

Figure 1. Table of current methods. (Forero et al., 2019; Koscianska et al., 2011; Li et al., 2009; Schwarzkopf et al., 2016; Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016).

Padlocks

Both rolling circle methods include padlock probes as part of their methodology. A padlock probe, which can be 30-150 nucleotides in length, is a single-stranded DNA (ssDNA) sequence designed to recognize a specific target sequence (see Fig. 2). The “arms” of a padlock probe are the ends of the ssDNA that are complementary to a specific target sequence (see Fig. 2). The middle sequence (the sequence between the arms) can be specifically designed to perform a function once amplified.

Figure 2. Diagram of a padlock probe.

Ligation

Some steps that are shared between RCA and RCT are the beginnings of hybridization and ligation. Hybridization is when the miRNA and the DNA attach, bringing the padlock arms close together. After hybridization, ligation occurs in which SplintR Ligase circularizes the miRNA. From here, RCT and RCA diverge.

SplintR Modification

Our team modified the protocols from the original paper used in their initial development. Instead of using T4 DNA ligase, we used SplintR Ligase as it “efficiently catalyzes the ligation of adjacent single-stranded DNA splinted by a complementary RNA strand” (Avantor Staff). Since SplintR ligase is specific to RNA-DNA hybridization, we chose to use SplintR to increase binding efficiency. As seen in Figure 3, SplintR works by utilizing phosphate modifications and ATP to ligate the DNA strand.

Figure 3. Animation of SplintR Ligase Function.

Padlock Design

For the padlock probe (PLP) design, part of the reverse complement of the miRNA makes up each end of the padlock probe. To determine where the reverse complement is split properly, we determined the annealing temperatures of each arm through SnapGene. To allow successful hybridization and maximize the binding efficiency of the miRNA and the padlock arms, the arms need to have the same annealing temperature. Furthermore, we added a phosphate group modification to the 5’ end of the padlock sequence to allow ligation by SplintR ligase (Jonstrup et al., 2006).

Figure 4. Padlock probe arrangement and formation.

Additionally, the alignment of the 5’ and 3’ ends is essential to determine where each part of the padlock + arms matches up to the target miRNA (Liu et al., 2013).As seen in Figure 4, the miRNA strand hybridizes antiparallel to the padlock arms. Therefore, the 5’ end of the miRNA will end up overlapping the 5’ PLP arm, and the 3’ end of the miRNA will end up overlapping the 3’ PLP arm (Liu et al., 2013).

The following are the steps for generating a padlock probe by hand. Researchers will need a software tool that displays melting temperatures of sequences such as the sequence generator SnapGene.

1. Paste your target biomarker in your software of choice.
2. Take the reverse complementary sequence of your target biomarker.
3. Split the sequence in half and put the second half in front of the first half to get the padlock arms.
4. Insert the desired reporter sequence in between the two arms.
5. Calculate the annealing temperature of both arms and move nucleotides one at a time from one end of an arm to the other end of the other arm until the difference between the annealing temperatures of the two arms is lowest.

Introduction to RCA/RCT

To optimize the miRNA point-of-care diagnostic tool, the team chose to experiment with both RCT (Rolling Circle Transcription) and RCA (Rolling Circle Amplification) to test their efficacy. Because they both use a similar approach, they share some of the same steps, including hybridization and ligation (Mohsen & Kool, 2016). The divergence in steps lies in the reporting mechanism where RCA utilizes fluorophore and quencher tagged linear DNA probes whereas RCT utilizes a broccoli aptamer (BBa_K3380153) (Filonov et al., 2014). Comparatively, the cost difference between using a linear DNA probe or an aptamer is negligible, and RCT and RCA provide straightforward outputs, making point-of-care diagnostics simple in both cases.

Overall, the rolling circle approach, with either RCT or RCA, provides great specificity for miRNA detection as it recognizes small nucleotide sequences and differences between types of miRNAs, as compared to SHERLOCK, toehold switches, and microarray analysis.

Therefore, the benefits of using one approach over the other are minimal, as both provide adequate approaches of measurement. However, researchers discovered RCT more recently than RCA, and less research exists on RCT’s usage for miRNA detection. As a result, we continued with RCT and RCA to test any differences between the two methods and their effectiveness in miRNA detection.

After experimentation, we have found that RCT was not successful in our lab. However, RCA has proved to create results that we can actually correlate to miRNA concentration. The predicted model made by our mathematical modeling team and the experimental curve tested by our RCA team matched up which proves that our biosensor mechanism does infact quantify miRNA.

RCT Design

Rolling Circle Transcription’s biosensor design utilizes padlock probes and Rolling Circle Transcription (RCT). When the target miRNA is present, the miRNA and the padlock probe arms form a DNA-RNA hybridization (see Fig. 5). Then, the introduction of SplintR ligase circularizes the padlock probe, allowing T7 RNA polymerase to attach and begin transcription (Wang et al., 2014). The product is a long, single-stranded RNA transcript that contains many copies of the reverse complement of the template strand, the padlock probe (Clausson et al., 2015).

Figure 5. Diagram showing how padlock probes can be combined with RCT for the detection of miRNAs (Wang et al., 2014).

Reporter Mechanisms

We chose the RNA fluorescent aptamer, Broccoli (BBa_K3380153), to be the reporter for the RCT reaction due to its relatively short length and excitation/emission wavelength similar to that of GFP (Filonov et al., 2014). We obtained the sequence of the aptamer through the iGEM parts registry and included the reverse complement of the sequence in the middle sequence of the padlock probes.

Spacer Regions

RCT yields long strands of repeating RNA sequences; to prevent the misfolding of the transcribed aptamers, we decided to include an upstream spacer sequence, a method inspired by the inclusion of a spacer sequence between two DNA aptamers for RCA conducted by researchers at the University of Arkansas (Al-Ogaili et al., 2020). The padlock spacer the team selected (BBa_K4245160) was derived from the Green Linker (Green, Silver, Collins, & Yin, 2014). We retrieved the Green Linker from the literature and manually modified it in NUPACK until there was no folding at our desired temperature of 25°C (Takahashi et. al, 2018). We then added this sequence upstream of the Broccoli complement sequence in our padlock probe.

RCT Discontinuation

Lambert iGEM attempted Rolling Circle Transcription but was unsuccessful. The products of RCT did not appear on a 1% agarose gel, shown in Figure 6, as it did with the RCA reaction gels, Figure 7, for example. To test if the secondary structure of the RCT padlock probe from the Broccoli aptamer complement was problematic to the transcription process by T7 RNA polymerase, we ran RCA with the RCT padlock probe. The results of a gel electrophoresis showed that RCA was successful with the RCT padlock probe, revealing that the folding of the RCT padlock probe is likely not the major problem (see Fig. 7). Considering the possibility that the RNA products may have degraded while loading and running RCT products on a gel, Lambert iGEM decided to continue testing RCT with the DFHBI-1T dye.

Figure 6. Picture of RCT products (circled in green) run on a 1% agarose gel. There were no visible bands that indicate the production of long RNA strands.

Figure 7. Picture of RCA gel products run with the miR-1 RCT padlock probe (circled in green). There were visible bands near the wells that indicate the production of long DNA strands.

To test the efficacy of the miRNA-Broccoli-spacer design incorporated into the RCT padlock probe, the sequence (BBa_K4245210) was transformed, cloned, mini-prepped, and tested in a cell-free reaction. As seen in Figure 8, the results showed a significant increase in fluorescence in reactions containing the miRNA-Broccoli-spacer coding plasmid, revealing the potential of the use of fluorescent aptamers through RCT. However, when we added DFHBI-1T, the fluorophore that the Broccoli aptamer uses to induce fluorescence, to the RCT products, there was no significant increase in fluorescence observed as compared to the controls (see Fig. 9).

Figure 8. Graph of fluorescence output of miR-Broccoli-spacer produced through cell-free Biobits. When we added the miR-Broccoli-spacer plasmid to the cell-free reaction, the fluorescence was significantly greater than without the plasmid.

Figure 9. Graph of fluorescence output before and after the addition of DFHBI-1T to RCT products and controls.

Therefore, Lambert iGEM discontinued the experimentation with RCT and focused on improving and testing RCA.

References