A New Method based on Peptide Mediated for Intracellular Linc-ROR Detection

Linc-ROR, as a new intergenic lncRNA, over-expressed in many kinds of cancer that promotes cancer cell proliferation. So, there are signicant values of Linc-ROR situ detectiction in diagnosis and research of diseases. While, the methods for LncRNA detection are almost outside the cells. In this study, established a method for Linc-ROR detection inside the cells. First, we put the nucleic acid probe and antibody together, probe entered cell by peptide (L17E) mediated endocytosis; In order to decreased background signals, the quenching probes were deliveried into cells to neutralise free uorescence probes. We veried the feasibility of this method, it has a huge amount of potential in clinical test.


Introduction
Long non-coding RNAs (lncRNAs) are a type of RNA, de ned as being transcripts with lengths exceeding 200nt that are not translated into protein 1,2 . 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 signi cance as a therapeutic target [3][4][5] .
At present, methods for the detection of RNAs include polymerase chain reaction (PCR), RNA-seq, gene chip or northern blotting 6-10 . 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 cells 12.13 . 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 di culties about intracellular linc-ROR detection. At rst, we need deliver detection uorescence probe (FP) to cells. While, nucleic acid FPs are negatively charged molecules, they cannot diffuse across cell membranes 14 . The FP deliver of traditional methods are assisted by liposome or nanomaterial [15][16][17] . Liposome is an arti cial vesicle composed of one or more concentric phospholipid bilayers and used especially to deliver microscopic substances to body cells 18 . In previous study, we found that although liposome could deliver FPs into cells, while the cytotoxicity and the transfer e ciency may warrant limiting its usefulness 19 . FPs mediation methods based on nanomaterials have developed rapidly in recent years because of their dual functions of quenching uorescence and carrying FPs 20,21 . However, nanomaterials are expensive, complicated, time-consuming and persistent cytotoxicity 22 . Our previous study found that FPs are closely bound to nanomaterials with low release e ciency, more than 40µg/mL of graphene oxide (GO) was needed to fully quench the 100nM uorescent-modi ed 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 micRNA 20,21 . 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 transport 23,24 . 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 nally be degraded in these compartments 25,26 . Misao Akishiba and his co-workers reported a new lipid-sensitive endosomolytic peptide named L17E, L17E had e cient endosomolytic activity that achieve a similar extent of endosomolytic activity with less of the peptide 27 . 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 modi ed 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 effect 28-30 and the uorescent signal would be quenched. Therefore, we can get the concentration of the target according to the uorescence intensity.

Extracellular Fluorescence detection
All oligonucleotide sequences and polypeptide were synthesized and purchased from Sangon Biotechnology Co. (Shanghai, China), and the sequences were provided in Table S1. Fluorescence signals were recorded using a Cary Eclipse Fluorescence spectrophotometer (Agilent, California).

Gel electrophoresis
The 10% BeyoGel™ Plus Precast PAGE Gel was purchased from Beyotime (Shanghai, China), and it was carried out at a constant voltage at 100V for 1h. Electrophoresis apparatus (DYY-6C, LIUYI, China) was used for the electrophoresis experiments. The gel was photographed by Bio-RAD digital imaging system.

Cellular uptake of FPAs and QPSAs
Breast cancer cells MDA-MB-231 were seeded into 96 well cell culture plate (NEST), and the cells were allowed to reach 80-90% con uence in 24h. These cells were washed twice with serum-free medium, and then incubated with 200nM probes with or without the presence of a peptide (10 µM) in serum-free medium for 2h at 37°C. Cells were washed with serum-free medium and incubated in serumsupplemented medium for 2 h at 37°C. The cellular uptake of probes was analysed in live cells using Axio Observer A1 inverted uorescence microscope.

Extracellular validation
Ideally, quenching probes (QPSAs) only react with free FPAs when enter into cells (Fig. 1B top). As shown in Fig. 1C, there is a strong uorescent signal when we added 200 nM FPAs to 100 nM linc-ROR targets (DFTs). Then, the uorescent signal among fell by close to 50 percent after super uous QP1 joined. The result proved that free FPAs were quenched. While, the uorescent signal had decreased dramatically after change QP1 to QPSA1. This suggested that when the bases were completely complementary, FPA/DFT would be competitive displaced by QPSA1 for more strong binding force (Fig. 1B middle). In order to solve this problem, we try to reduce the complementary base pairs between FPA and QPSA. The experiment results shown that QPSA3 with sixteen pairs of complementary bases can reach a balanced change (Fig. 1B bottom). So, we chose QPSA3 as the optimum condition for the following research.

Intracellular validation
The strategy is illustrated in Fig. 2A. The lytic activity of L17E was weaker, because of a negatively charged Glu residue was inserted in the hydrophobic face. The net positive charges of the peptide can help its endocytosis uptake and cell surface adsorption ( Figure S1). FPAs were delivered into the cells through L17Es mediated endocytosis. Then, the peptide preferentially perturbs the endosomal membranes to attain the cytosolic release of FPAs with su cient e cacy to Htarget detection.
Next, we evaluated the ability of these peptides to promote the take in and cytosolic release of endocytosed biomacromolecules. All the results shown in Fig. 2B. MDA-MB-231 cells were incubated with probes and L17Es in medium for two hours, and the cellular distribution of the uorescence signal was analysed using Axio Observer A1 inverted uorescence microscope. On the other hand, when cells were treated with FPAs in the presence of L17E (10 µM), a signi cant expression of uorescence signal was observed in the cells (Fig. 2B bottom), which suggests that the L17E-mediated delivery of FPAs into cells.
In contrast, no signi cant uorescence signals were observed after treatment with FPAs in the absence of L17E (Fig. 2B top). In addition, the experimental results shown that only nucleic acid probes catenating with antibodies can be delivered to cells. These results collectively indicate that L17E allows the delivery of FPAs into the cytosol.

Cytosolic delivery of QPSA using L17E
Free FPAs without hybridization with linc-ROR targets were background signal. In this study, we innovation used a kind of quenching probe which bound with anti-mouse secondary antibody (QPSA) to decrease background signal. The QPSAs were labeled with Dabcyl which could quench the uorescence of FAM when they were closed. The QPs and secondary antibodies were forming QPSAs at the same way. Then, the QPSAs were delivered into the cells with the same method (Fig. 3A). Applying the principle of speci c binding of antibody and secondary antibody, the FPs and QPs were closed through proximity ligation assay and the uorescence were quenched. While, antibody-DNA probes hybridization with linc-ROR would not be quenched and keep a strong uorescence. compared to the control group (Fig. 3B top), the uorescence signal could not change when QPSAs incubated with cells in the absence of L17Es (Fig. 3B middle). While, the uorescence signal signi cant declined in the absence of L17Es (Fig. 3B bottom). All results veri ed that QPSAs could be delivered to cells and free FPAs would be quenched.

Verify in other tumor cell lines
Then, the proposed strategy was used to estimate the intracellular expression levels of Linc-ROR in different living cancer cells, breast cancer cell BT-549 and adrenocortical cell HAC15. As shown in Fig. 4, as for MDA-MB-231 and BT-549 cells, two breast tumor cell lines, we could observe bright FPA uorescence images. Also, the uorescence intensity of FPA in BT-549 cells was stronger than MDA-MB-231 cells. While, the uorescence intensity of HAC15 cells was very week. The results were in keeping with Linc-ROR RNA Q-PCR results (Figsure S2).

Conclusions
In this report, we have demonstrated a new detection strategy for Linc-ROR in cells. First, we linked the nucleic acid probe and antibody together. Then, L17E a single negatively charged Glu residue into the potentially hydrophobic face of M-lycotoxin was used to delivery probes into cells through endocytosis. In order to decreased background signals, the quenching probes were deliveried into cells at the same way. At last, we verify the feasibility of this method, it has a huge amount of potential in clinical test.
Furthermore, our approach can also be potentially applied for detect of other RNAs in cells.

Notes
The authors declare no competing nancial interest.

Supplementary Files
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