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.
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.
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.