Part:BBa_K4683002

hsa-miR-1-3p eRCA Padlock Probe

This part is the sequence for the hsa-miR-1-3p eRCA Padlock Probe, which our team designed to be specific to BBa_K4245006, hsa-miR-1-3p. This miRNA acts as a biomarker for Coronary Artery Disease and is therefore potentially useful for the early detection of this condition. Contrary to typical Rolling Circle Amplification (RCA) padlock probes, besides the 3’arm, BBa_K4245100 and 5’ arm BBa_K4245107, the eRCA probe consists of an additional part between the arms and the middle sequence: Nb.BbvCI nicking enzyme cut sites BBa_M31961 (Li et al., 2017). Nicking endonuclease, Nb.BbvCI, is then able to recognize these sites during amplification and cleave only one strand of the eRCP (exponential rolling circle product) while it is still bound to the padlock probe (Biolabs) This ensures that the padlock probe remains intact while releasing individual strands of the target miRNA and middle sequence. These miRNAs 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. The produced transcript contains cut repeats of the middle sequence (see fig.). In this case, the complement of the Lettuce Aptamer BBa_K4683000 was used so that during the amplification process, Lettuce Aptamers would be produced.

Figure. Modified exponential rolling circle amplification process

References

Biolabs, N. E. (n.d.). Nb.BbvCI. NEB. https://www.neb.com/products/r0631-nbbbvci#Product%20Information

Li, X.-Y., Du, Y.-C., Zhang, Y.-P., & Kong, D.-M. (2017). Dual functional phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Scientific Reports,7(1). https://doi.org/10.1038/s41598-017-06594-1
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]