Theranostics 2020; 10(10):4410-4421. doi:10.7150/thno.42951

Research Paper

Nondestructive analysis of tumor-associated membrane protein MUC1 in living cells based on dual-terminal amplification of a DNA ternary complex

Xiaohao Liu1*, Dongsheng Mao1*, Guoli Deng2, Yuchen Song1, Fan Zhang1, Shiqi Yang1, Genxi Li1, Feng Liu3✉, Wei Cao4✉, Xiaoli Zhu1✉

1. Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China
2. Plant Science Center, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China
3. Oncology Department, Shanghai Ninth People's Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 201900, P. R. China
4. Department of Oral and Maxillofacial-Head and Neck Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, P. R. China
*These authors contributed equally in this work

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Citation:
Liu X, Mao D, Deng G, Song Y, Zhang F, Yang S, Li G, Liu F, Cao W, Zhu X. Nondestructive analysis of tumor-associated membrane protein MUC1 in living cells based on dual-terminal amplification of a DNA ternary complex. Theranostics 2020; 10(10):4410-4421. doi:10.7150/thno.42951. Available from http://www.thno.org/v10p4410.htm

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Abstract

Non-destructive analysis of cells at the molecular level is of critical importance for cell research. At present, immunoassay-based and aptamer-based methods can achieve non-structural destructive cell analysis, but still lead to changes in cells at the molecular level. Here, we have proposed a dual-terminal amplification (DTA) strategy, which enables nondestructive analysis of membrane protein MUC1 without the effect on protein expression and cell viability in living cells.

Methods: A fluorophore (Cy5)-labeled DNA ternary complex consisting of three oligonucleotides is designed. It can recognize MUC1 through its aptamer region, and thus make the MUC1 of cells visible under a fluorescence microscope. When DNA polymerase is added, dual-terminal amplification is performed. One direction dissociates aptamer from MUC1, and the other direction, also known as rolling circle amplification (RCA), produces long linear DNA strands, which can be further adopted for quantitative analysis of MUC1. In this way, all reagents are removed from the surface of the cells after the analysis, which allows nondestructive analysis. We named this strategy dual-terminal amplification (DTA) analysis.

Results: By using the DTA analysis, both in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 were achieved. In addition, the aptamer-containing DNA ternary complex stays on cell surface only during the analysis and leaves the cell after the analysis is complete. The cells can be maintained in a non-interfering state for the rest of the time. So after the analysis, it is found that there are no effect on the physiological activity of cells and the expression of target protein even after two rounds of repeatable imaging and quantitative analysis.

Conclusion: In summary, we have successfully constructed a strategy for nondestructive analysis of membrane protein in living cells. We believe that this method provides a promising way for the analysis of the key membrane proteins of cells and the versatile utilization of precious cell samples.

Keywords: membrane protein, dual-terminal amplification, nondestructive, in situ fluorescence imaging, quantification