BBa_K4664002: Padlock-223


The padlock probe is essential to our experiments, especially the ligation and rolling circle amplification (RCA) steps. Both of these steps are included in the intermediary assays and our final MB-ERC2 (miRNA biomarker-based exponential RCP Cas12a/CRISPR) system. The general MB-ERC2 schematics is shown below in Figure 1.

Figure 1. MB-ERC2 Reaction Schematics.


Padlock-223 is a 61 bp-long linear single-stranded DNA (ssDNA). It is composed of three sections: the miR-223 binding site (ligation module), crRNA recognition site (Cas12a detection module), and the intermediate sequence. Figure 2 is a visualisation of the padlock probe construct.

Figure 2. Padlock Probe Basic Construct

To optimise the Padlock-223 sequence, we performed experiments with single, double, and triple ligation module mismatches and alternating Cas12a detection module locations (Parts BBa_K4664007 to BBa_K4664023). For results of the optimization experiments, please see registry pages for BBa_K4664007 and BBa_K4664010.

We also performed a series of experiments, aiming to optimise our MB-ERC2 reaction system. These include experimenting with different concentrations of the various reagents used in the assay, trying out different reagents/buffers and buffer concentrations, and the role of RCA amplicon in secondary RCA initiation. More information on these could be found on our Experiments page.

The Cas12a detection module sequence is identical to the crRNA (aside from the difference in T and U). This is due to the crRNA’s complementarity to the RCA amplicon (product of DNA synthesis with the padlock as template). More information on crRNA could be found on the registry page for BBa_K4664005.



The webpage at presents a comprehensive set of results derived from a series of experiments focused on Padlock-223. This includes detailed data from intermediary assays, both the three-step and two-step processes. In addition, the page showcases outcomes from our experiments involving MB-ERC2, as well as a collection of optimization trials conducted to enhance the overall process.


Padlock-223 and the MB-ERC2 system represent significant progress in terms of COPD detection. The verified LOD is 20 fM, which is comparable to RT-qPCR, the ‘golden standard’ for miRNA detection (for our RT-qPCR results, please see BBa_K4664000). Moreover, reaction time was shortened to 20-100 minutes, and cost was cut down to $1 per reaction for reagents (see Contribution page). We also believe that the MB-ERC2 system is potentially applicable for the detection of other diseases with miRNA biomarkers.


Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection method using SplintR Ligase. Nucleic Acids Research, 44(13), e116.

He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering.