Part Collection

Parts Collection

To address a scarcity of research regarding the correlation of various microRNAs (miRNAs) with coronary artery disease (CAD), we introduced a collection of 17 new parts that work with rolling circle amplification (RCA) to detect miRNAs (see RCA). To offer flexibility for future RCA applications, we also designed two different reporter mechanisms: linear DNA probes and the DNA aptamer Lettuce. Linear probes rely on two sequences (fluorophore and quencher) that hybridize to rolling circle products (RCP). When bound in proximity, the quencher sequence absorbs the energy from the fluorophore which results in a detectable decrease in fluorescence. For the Lettuce reporter, an increase in RCP results in an increase in fluorescence. By utilizing RCA reactions with two different customizable reporters, we aim to increase feasibility of miRNA research in low resource areas.

A padlock probe, approximately 30-150 nucleotides in length, is a single-stranded DNA sequence designed to recognize a specific biomarker sequence. The “arms” or ends of the padlock probe are complementary to a specific target sequence. The middle sequence between the arms performs a specific function when amplified. Once the target sequence binds to the padlock probe, the biomarker serves as a primer to initiate RCA (see Fig. 1). Lambert iGEM applied these mechanisms to provide a potential diagnostic tool for CAD, using miRNA 1, 133a, and 451a as the biomarkers.

Figure 1. Diagram of a padlock probe, the “arms” or ends of the padlock probe are complementary to a specific target sequence.

• Each pair of arms – BBa_K4245100 and BBa_K4245103, BBa_K4245106 and BBa_K4245107, BBa_K4245110 and BBa_K4245113 – is complementary to miRNA 1 (BBa_K4245006), 133a (BBa_K4245009), and 451a (BBa_K4245012), respectively.

• The fluorophore (BBa_K4245130) and quencher (BBa_K4245132) bind to the middle sequence of the padlock probes in a head-to-head alignment, which allows the quencher to suppress the natural fluorescence emitted by the fluorophore.


Figure 2. Production of repeated linear DNA probes binding sites by RCA in the presence of target miRNA.




• BBa_K4245134 and BBa_K4245135 are two halves of the whole Lettuce aptamer that are each complementary to a portion of the padlock probe. When they bind to the padlock probe, they form the full Lettuce aptamer that can produce fluorescence in the presence of DFHBI-1T.


Figure 3. Diagram of the Lettuce aptamer.




• We constructed the composite padlock probes – BBa_K4245200, BBa_K4245204, BBa_K4245209 – using the pair of arms corresponding to the specific miRNA targets and BBa_K4245131 as the middle sequence.

These parts work together to make an effective and powerful detection method for miRNAs 1, 133a, and 451a. We hope these parts can empower scientists and healthcare providers to better understand the correlation between miRNAs and CAD.