In our project, we have developed biosensors based on toehold switches for sequence-based detection of PANTR1, a long non coding (lnc) RNA [1] overexpressed in different types of cancer cells that was shown to promote cell migration, invasion, angiogenesis and proliferation and to repress apoptosis [2–9].

We employed established computational approaches [10] to design and optimise the specificity of our biosensor towards the four variants of the PANTR1 lncRNA:

• PANTR1 v1 (GenBank Acc. N° NR_037883): 368 toehold switches designed
• PANTR1 v2 (GenBank Acc. N° NR_131233): 38 toehold switches designed
• PANTR1 v3 (GenBank Acc. N° NR_131234): 35 toehold switches designed
• PANTR1 v4 (GenBank Acc. N° NR_131235): 44 toehold switches designed

A total of 396 distinct toehold switches were designed for the PANTR1 lncRNA. The full list and and their parameters is available as an excel file.
Among them, we selected the top 3 best ranked toehold switches and their cognate triggers, and cloned them with a promoter and a terminator, in order to characterise them in vivo in E. coli.

Toehold Switch Label	Sensor Part Number	Sensor + sfGFP-LVAtag Part Number	Trigger Part Number	Trigger Transcriptional Unit Part Number
PANTR1 Toehold Switch n°1	BBa_K4432001	BBa_K4432101	BBa_K4432201	BBa_K4432301
PANTR1 Toehold Switch n°2	BBa_K4432002	BBa_K4432102	BBa_K4432202	BBa_K4432302
PANTR1 Toehold Switch n°3	BBa_K4432003	BBa_K4432103	BBa_K4432203	BBa_K4432303
PANTR1 Toehold Switch n°4	BBa_K4432004	BBa_K4432104	BBa_K4432204	BBa_K4432304
PANTR1 Toehold Switch n°5	BBa_K4432005	BBa_K4432105	BBa_K4432205	BBa_K4432305
PANTR1 Toehold Switch n°6	BBa_K4432006	BBa_K4432106	BBa_K4432206	BBa_K4432306
PANTR1 Toehold Switch n°7	BBa_K4432007	BBa_K4432107	BBa_K4432207	BBa_K4432307
			BBa_K4432407	BBa_K4432507
			BBa_K4432607	BBa_K4432707
PANTR1 Toehold Switch n°8	BBa_K4432008	BBa_K4432108	BBa_K4432208	BBa_K4432308

References

[1] Vance KW, Ponting CP. Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends in genetics: TIG (2014) 30: 348–355.
[2] Li W, Zheng J, Deng J, You Y, Wu H, Li N, Lu J, Zhou Y. Increased levels of the long intergenic non-protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells. Gastroenterology (2014) 146: 1714-1726.e5.
[3] Guo H, Wu L, Yang Q, Ye M, Zhu X. Functional linc-POU3F3 is overexpressed and contributes to tumorigenesis in glioma. Gene (2015) 554: 114–119.
[4] Tong Y-S, Wang X-W, Zhou X-L, Liu Z-H, Yang T-X, Shi W-H, Xie H-W, Lv J, Wu Q-Q, Cao X-F. Identification of the long non-coding RNA POU3F3 in plasma as a novel biomarker for diagnosis of esophageal squamous cell carcinoma. Molecular Cancer (2015) 14: 3.
[5] Shan T-D, Xu J-H, Yu T, Li J-Y, Zhao L-N, Ouyang H, Luo S, Lu X-J, Huang C-Z, Lan Q-S, Zhong W, Chen Q-K. Knockdown of linc-POU3F3 suppresses the proliferation, apoptosis, and migration resistance of colorectal cancer. Oncotarget (2016) 7: 961–975.
[6] Lang H-L, Hu G-W, Chen Y, Liu Y, Tu W, Lu Y-M, Wu L, Xu G-H. Glioma cells promote angiogenesis through the release of exosomes containing long non-coding RNA POU3F3. European Review for Medical and Pharmacological Sciences (2017) 21: 959–972.
[7] Li Y, Li Y, Wang D, Meng Q. Linc-POU3F3 is overexpressed in hepatocellular carcinoma and regulates cell proliferation, migration and invasion. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie (2018) 105: 683–689.
[8] Wan X, Xiang J, Zhang Q, Bian C. Long noncoding RNA POU3F3 promotes cancer cell proliferation in prostate carcinoma by upregulating rho-associated protein kinase 1. Journal of Cellular Biochemistry (2019) 120: 8195–8200.
[9] Li W, Wu X, She W. LncRNA POU3F3 promotes cancer cell migration and invasion in nasopharyngeal carcinoma by up-regulating TGF-β1. Bioscience Reports (2019) 39: BSR20181632.
[10] To AC-Y, Chu DH-T, Wang AR, Li FC-Y, Chiu AW-O, Gao DY, Choi CHJ, Kong S-K, Chan T-F, Chan K-M, Yip KY. A comprehensive web tool for toehold switch design. Bioinformatics (Oxford, England) (2018) 34: 2862–2864.