Long non-coding RNAs (lncRNAs) are a type of RNA, defined as being transcripts with lengths exceeding 200nt that are not translated into protein1,2. Linc-ROR, as a new intergenic lncRNA, over-expressed in many kinds of cancer that promotes cancer cell proliferation, including breast cancer (BC), pancreatic cancer (PC), colorectal cancer (CRC) and so forth. As linc-ROR can regulate cell proliferation, apoptosis, migration, and invasion, it can thus be used as a potential biomarker for patients with tumors and has potential clinical significance as a therapeutic target3–5.
At present, methods for the detection of RNAs include polymerase chain reaction (PCR), RNA-seq, gene chip or northern blotting6–10. Although these methods do have their own advantages, but some drawbacks limit their use in clinical detection. Firstly, these methods involve extracting RNAs from cells, while linc-ROR are not stable and easily degraded by Ribonuclease R (RNase R)11. Then, current methods can only obtain expression quantity of lncRNAs, but ignore the position of lncRNAs in different cells12.13. Therefore, development of rapid, simple, sensitive diagnostic systems for intracellular linc-ROR detection is emerging as a necessary response to develop of clinical laboratory diagnosis and life science research.
There are several difficulties about intracellular linc-ROR detection. At first, we need deliver detection fluorescence probe (FP) to cells. While, nucleic acid FPs are negatively charged molecules, they cannot diffuse across cell membranes14. The FP deliver of traditional methods are assisted by liposome or nanomaterial15–17. Liposome is an artificial vesicle composed of one or more concentric phospholipid bilayers and used especially to deliver microscopic substances to body cells18. In previous study, we found that although liposome could deliver FPs into cells, while the cytotoxicity and the transfer efficiency may warrant limiting its usefulness19. FPs mediation methods based on nanomaterials have developed rapidly in recent years because of their dual functions of quenching fluorescence and carrying FPs20,21. However, nanomaterials are expensive, complicated, time-consuming and persistent cytotoxicity22. Our previous study found that FPs are closely bound to nanomaterials with low release efficiency, more than 40µg/mL of graphene oxide (GO) was needed to fully quench the 100nM fluorescent-modified single-stranded nucleic acid probe. Because of lncRNA are much longer than micRNA, these methods are not suitable for lncRNA detection. they are mainly using for detection of micRNA20,21. Endocytosis is a cellular process in which substances are brought into the cell. The material to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material. It is a form of active transport23,24. So endocytosis is a simple and rapid process without any damage to cells, which is an ideal way of FPs delivery. Antibodies and other biological macromolecules can enter cells through endocytosis easily. In this study, we combined FP with antibody and introduce it into cell through endocytosis.
While, one of the main barriers of this method is the limited release of FPAs from endosomes into the cytosol. FPAs are taken up by cells via endocytoses, which is a process that involves the physiological uptake of extracellular substances delivered into cells by encapsulation into vesicular compartments named endosomes. Without their release from vesicular compartments into the cytosol, FPAs cannot reaction with their target molecules, and may finally be degraded in these compartments25,26. Misao Akishiba and his co-workers reported a new lipid-sensitive endosomolytic peptide named L17E, L17E had efficient endosomolytic activity that achieve a similar extent of endosomolytic activity with less of the peptide27. So, we used lipid-sensitive polypeptide L17E to deliver FPAs in to cells.
In addition, uncombined FPAs are able to increase background signals without restrictive measures. So, one kind of quenching probe (QP) was designed to combine free FPA and reduce the background signal. The QP was modified with a secondary antibody (QPSA) which could bind to the antibody on the FP. After the QPs were delivered into the cells with the same manner, the antibody on the FPs and the secondary antibody on the QPs would band together. If the FPAs were free, they would rapidly hybridize with the QPSAs through the proximity ligation effect28–30 and the fluorescent signal would be quenched. Therefore, we can get the concentration of the target according to the fluorescence intensity.