Part:BBa_K3730012:Design

miRNA sponge, to reduce specific types of free hsa-miR-199a, 195 and 22 level in cell

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 289
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 289
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 289
  Illegal BamHI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 289
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 289
  
• 1000
  COMPATIBLE WITH RFC[1000]

          Fig. 1 The sequence of miRNA sponge.

The sponge is composed of several complementary tandem repeats of each miRNA flanking by two restriction enzyme sites.

Design Notes

The secondary structure and miRNA binding affinity of sponge were evaluated by RNA fold web server and miRNAsong to make sure the miRNA binding sites were exposed and it had a strong attraction to free miRNA which was illustrated in Fig. 2 and Fig. 3.

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  Fig. 2 miRNA sponge secondary structure prediction.

The green paired bases had relatively low binding affinity which would not affect the binding of miRNA while the red bases had tight binding. Both possible linear and circular structures were shown above.

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 Fig. 3 The affinity and kinetics of binding of each miRNA to its complementary sequence in miRNA sponge.

It indicated that our miRNA sponge was likely to absorb the free miRNA in the cytosol.

Source

NO

References

[1] Kluiver, J., Slezak-Prochazka, I., Smigielska-Czepiel, K., Halsema, N., Kroesen, B. J., & van den Berg, A. (2012). Generation of miRNA sponge constructs. Methods, 58(2), 113-117.

[2] Barta, T., Peskova, L., & Hampl, A. (2016). miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico. Scientific reports, 6(1), 1-8.