Part:BBa_K2908666

miR148b-sponge

This part is designed by miRNASong(an online software) and synthesized by company.It is used to down-regulate the level of miR148b in heterogenous TNBC cells.

Figure1. Picture A shows the sequence of this part; The heat map shows as (B).

Construction
Firstly, obtain the basic sequence information of miRNA from miRBASE, analyze which miRNA subtype is specifically described according to the literature table provided, obtain the sequence and identify the seed sequence.[1] The miRNAsong website[2] was used to design the basic sponge framework, the free energy cutoff was 25Kcal/mol, and the miss was checked by 2-8seed classic matching. Since more than one BS will be connected in the following experiments, spacer connection is directly designed between two BS in the design stage to facilitate the judgment of the overall change after spacer connection. Then manually annotate the mismatch site, spacer sequence. The website does not consider the off-target effect at all, and most of the results are so severe that the 9-12 base combination needs to be manually changed according to the site. Get the combination with lower miss, perform secondary structure verification, and record the passed combination.

Figure2. This figure demonstrates that when combining the sponge into a test plasmid, miR148b-sponge can efficiently inhibit the endogenous miR148b compared to miR141-sponge when driven by the TNBC-specific promoter s(ESR1)p.

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]

References:
[1] Ebert, M.S., and Sharp, P.A. (2010). MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050.
[2] Tomas Barta, Lucie Peskova & Ales Hampl, (2018), miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico, Scientific Reports.