Part:BBa_K2624006:Design
MAP4K4 miRNA sponge
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 178
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 178
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 178
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Ebert et al. once designed microRNA sponge to down-regulate the number of mature miRNA in 2007. MiRNA sponge causes the disinhibition of target mRNA as its competitive inhibitor. It is an important tool to study the function of miRNA, and has potential getting involved in crucial therapeutics of some disease. An optimal sponge should accord with these features:(1) 6-9 microRNA binding sites (MBS); (2) a central bulge to avoid the degradation of the sponge, this would be realized by the addition, elimination or substitution of some nucleotides; (3) a four-nucleotide spacer; (4) a strong promoter.
The target sequence is 5’-UAAGAGGAUAGACCUUCCC-3’. The relative contribution of the four bases A, G, U, C, leads to self-complementary base pairing, which adds difficulty the sponge design. In order to solve this problem, MFold (http://unafold.rna.albany.edu/?q=mfold) was used to predict the secondary structure and ΔG of the designed sponge, and RNA Structure (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/PredictBi/PredictBi.html) to evaluate the interaction between the sponge and microRNA. Based on the principal of “molecules with lower energy are more stable”, we chose the sponge with a low ΔG when it’s alone and a high ΔG when it interact with the miRNA(MAP4K4). For stable interaction between the sponge and miRNA, the bulge was at the central position of the sponge.
For stable interaction between sponge and miRNA, we had 6 MBSs in the sponge, and the bulge at the center of the sponge. However, there are 49 nucleotides before the sponge and 10 nucleotides after it which are present due to practical reasons but not relevant for miRNA absorption. We suggest future teams to delete them.
Source
Non.
References
[1]Ebert M S, Neilson J R, Sharp P A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells[J]. Nature methods, 2007, 4(9): 721.
[2]Ebert M S, Sharp P A. MicroRNA sponges: progress and possibilities[J]. Rna, 2010.
[3]de Melo Maia B, Ling H, Monroig P, et al. Design of a miRNA sponge for the miR-17 miRNA family as a therapeutic strategy against vulvar carcinoma[J]. Molecular and cellular probes, 2015, 29(6): 420-426.