Part:BBa_K4664009

Padlock-miR-223-4

Padlock Padlock-miR-223 is the corresponding padlock chosen for the miR-223 biomarker.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Background

In order to determine the PLP sequence that yields the best reaction kinetics, we designed padlocks with the crRNA recognition site (responsible for binding with the crRNA in RNP and activating Cas12a cleavages) at different locations.

Design

In Padlock-miR-223-4（Padlock-4）, the Cas12a detection module (square brackets) is placed in the middle.
Sequence:
TCAAATACACG-AGATACCCT-[CGTCGCCGTCCAGCTCGACC]-AACCATCGAT-AACTCAGCTTG

Results

The following figure shows the final values for padlocks 1-4 (padlock 1 is BBa_K4664002, aka. Padlock-223).

Figure 1. Padlocks 1-4.

References

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. https://doi.org/10.1093/nar/gkw399
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. https://doi.org/10.1038/s41551-023-01033-1