Part:BBa_K4438121

FASTmiR-222-D2_s

FASTmiR stands for Fluorescence Aptamer Sensor For Tracking miRNAs. It is an RNA-based sensor for in-vitro quantification of miRNAs. This part is the expression cassette for BBa_K4438101 with BBa_J64997 upstream and BBa_K731721 downstream along with some spacer sequences BBa_K3831027 (at 5’ end) and BBa_K4438136 (at 3’ end). FASTmiR-222-D2_s, after transcription, binds to miR-222, the transcription product of BBa_K4438150. The binding of these to RNAs will lead to the revelation of a binding site for DFHBI, a fluorophore, where it gets trapped and shows fluorescence.

Usage and Biology

This part can be used for the detection of miR-222, which proves to be a biomarker for many diseases. A promoter sequence added in the beginning enhances its usage in both in-vivo and in-vitro conditions. The OFF(A) and ON(A) structure for the sensor was generated as given: We performed an In-silico analysis where we replaced the 12th position A with G and the 86th position U with C in Spinach 2.1 to generate a modified Spinach 2.6 PDB file. We used this to confirm the decrease in the binding energy of aptamer and dye.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 100
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 176
  Illegal SpeI site found at 100
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 100
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 100
  
• 1000
  COMPATIBLE WITH RFC[1000]

Notes

The split position of the complementary sequence in the sensor is ACCCAGTAGCC....AGAGTGAGCT

References

Huang, K., Doyle, F., Wurz, Z. E., Tenenbaum, S. A., Hammond, R. K., Caplan, J. L., & Meyers, B. C. (2017). FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs. Nucleic acids research, 45(14), e130-e130