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.

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