Team:SFLS Shenzhen/Design
Project Design
Small molecule miRNA is a class of endogenous single-stranded non-coding small RNAs of 21-24 nucleotides in length. miRNA not only plays an important role in the normal development, differentiation and apoptosis of the body, but also participates in the occurrence of various tumors including breast cancer, and regulates the occurrence and development of breast cancer at the microscopic level.
Serum MIRNA can be used as a sensitive marker for breast cancer, and its sensitivity and specificity are higher than the currently widely used carbohydrate antigens. [4] Studies have found that compared with healthy controls, miR-10b, miRNA-145 and miR-155 are up-regulated in the serum of breast cancer patients, and the expression levels of the three miRNAs are significantly correlated with tumor metastasis, but they have no correlation with age, histological features, and ER expression.
Small molecule miRNA is a class of endogenous single-stranded non-coding small RNAs of 21-24 nucleotides in length. miRNA not only plays an important role in the normal development, differentiation and apoptosis of the body, but also participates in the occurrence of various tumors including breast cancer, and regulates the occurrence and development of breast cancer at the microscopic level.
Serum MIRNA can be used as a sensitive marker for breast cancer, and its sensitivity and specificity are higher than the currently widely used carbohydrate antigens. [1] Studies have found that compared with healthy controls, miR-10b, miRNA-145 and miR-155 are up-regulated in the serum of breast cancer patients, and the expression levels of the three miRNAs are significantly correlated with tumor metastasis, but they have no correlation with age, histological features, and ER expression.
Based on the sensitivity and specificity of microRNAs in serum, we use the Toehold switches, a tool for detecting MIRNA in synthetic biology, to construct a joint detection of miR-10b and miR-155 by constructing our gene loop.
Toehold switch is a strain of RNA which has a hairpin loop that locks the ribosome to prevent it from further transcription. When another strain of RNA(called the trigger) matches with the switch, the hairpin loop unlocks itself and the transcription begins.[2]
Most toehold switch triggers are over 30bp long so they can match both the trigger and the stem. However, miR-155 is only 24bp long and miR-10b is 23bp long. To ensure a successful detection, we cut the stem below the start codon 1bp shorter to match miR-155 and 2bp shorter for miR-10b.
Each trigger is used for matching a certain type of miRNA and start the transcription.In Toehold switch designing,a Switch will act as a “lock” while a Trigger(miRNA) will act as a “key”.The “lock” will only be unlocked by a certain type pf “key”.We designed our switch in order to match miRNA-155 and miRNA- 10b and “unlock” gene expression.
Trigger A(miRNA-155)
UUAAUGCUAAUCGUGAUAGGGGUU
Switch A:
GGGAACCCCUAUCACGAUUAGCAUUAAGAGAUAUAUATTAAAGAGGAGAAAAUAUAUAUGUUAAUGCUAAACCUGGCGGCAGCGCAAAAG
Trigget B（miRNA-10b）
UACCCUGUAGAACCGAAUUUGUG
Switch B:
GGGCACAAAUUCGGUUCUACAGGGUAGAGAUAUAUATTAAAGAGGAGAAAAUAUAUAUGUACCCUGUAAACCUGGCGGCAGCGCAAAAG
switch a
switch b
In order to be utterly sure that a person has breast cancer, we intended to design a circuit which only express GFP when both these miRNAs are detected. AND Gate design appears to be important.The picture below shows our final design.
the AND gate
Our toehold switch for miR-155(Switch A) is followed by a sequence of protein ECF41_491, which, after being transcribed and translated, will release the protein and activate promoter P41_up1141 and leads to the transcription to Trigger C. As for Switch B, it is followed by the sequence of protein ECF42_44454, which will activate promoter P42_up4062 and start the transcrption of Switch C. When Trigger C matches with Switch C, it will start the translation of GFP.[2]
In conclusion,our circuit will only express GFP when both miRNAs are detected.Based on this,we can detect fluorescence concentration and use math model to calculate miRNA expression level .
References
- 1,陆婉玲 王志刚.血清小分子RNA与乳腺癌关系研究进展。中国医药，2013，8（11）；1677-1679
- 2,Toehold Switches——De-Novo-Designed Regulators of Gene Expression