Detection of intra-brain cytoplasmic 1 (BC1) long noncoding RNA using graphene oxide-fluorescence beacon detector

Detection of cellular expression of long noncoding RNAs (lncRNAs) was elusive due to the ambiguity of exposure of their reactive sequences associated with their secondary/tertiary structures and dynamic binding of proteins around lncRNAs. Herein, we developed graphene-based detection techniques exploiting the quenching capability of graphene oxide (GO) flakes for fluorescent dye (FAM)-labeled single-stranded siRNAs and consequent un-quenching by their detachment from GO by matching lncRNAs. A brain cytoplasmic 1 (BC1) lncRNA expression was significantly decreased by a siRNA, siBC1–1. GO quenched the FAM-labeled siBC1–1 peptide nucleic acid (PNA) probe, and this quenching was recovered by BC1. While FAM-siBC1–1-PNA-GO complex transfected spontaneously mouse or human neural stem cells, fluorescence was recovered only in mouse cells having high BC1 expression. Fluorescent dye-labeled single-stranded RNA-GO probe could detect the reactive exposed nucleic acid sequence of a cytoplasmic lncRNA expressing in the cytoplasm, which strategy can be used as a detection method of lncRNA expression.

Here, we developed the sensing techniques for the expression of BC1 lncRNA via siRNA-based probe selection. Specific probe to detect BC1 lncRNA was selected via siRNA-based knockdown test for BC1 lncRNA. GO that has relatively homogenous size was chosen as reliable carrier in GO-based lncRNA sensor (Fig. 1). We could delineate the efficacy of siPNA probe-GO system to sense intracytoplasmic BC1 lncRNA expression by signal-on way in live neural stem cells in vitro.

Results
Design of siRNA-based probe for BC1 lncRNA detection. The presence or absence of lncRNAs such as BC1 was easily examined using fractionated organelle qRT-PCR but the active functioning of the lncRNA in the intracellular milieu can only be observed by molecular imaging using fluorescent proteins, sometimes transduced and persistently expressing or in other times transiently transfected to the cells. The four siRNAs for BC1 lncRNA were designed to correspond to the nucleotides 98-116 (siBC1-1), 110-128 (siBC1-2), 127-144 (siBC1-3) and 136-154 (siBC1-4) (Fig. 2a). The siBC1-1 and siBC1-2 recognized the loop region of BC1 lncRNA, whereas siBC1-3 and siBC1-4 targeted the spanned stem region. These four siRNAs were transfected into the mouse neural stem cells NE-4C or the mouse lung adenoma LA-4 cells. The BC1 lncRNA expression decreased by siBC1-1 or siBC1-2 in comparison to the siNC in NE-4C cells (Fig. 2b, n = 3). In LA-4 cells, the BC1 lncRNA expression also decreased by siBC1-1 and siBC1-2 ( Fig. 2c, n = 3). The siRNA-mediated reduction of BC1 lncRNA transcripts revealed siBC1-1 as the best candidate. We have selected another siBC1-3 to assess the difference of the probe by the efficiency of the siRNA. Each siRNA probe was modified as a PNA form to enhance the stability and sequence of PNA probe for BC1 lncRNA is in table supplement. As controls, we used the scrambled form of FAM-PNA-BC1-1 (FAM-PNA-scr1). Size was determined using a Nanoparticle Tracking Analysis after FAM-PNA-BC1-1 probe was loaded with GO sheet. The overall size distribution and maximum size peak between GO sheet and FAM-PNA-BC1-1-GO were not changed (Supplementary Figure S1).

Specificity of FAM-PNA-GO complex.
To find the optimal concentration of GO to maximize the fluorescence quenching efficacy, varying concentration of GO was mixed with FAM-PNA probes (40 pmoles), showing that 0.4 μg of GO completely quenched the fluorescence (Fig. 3a,b). Upon addition of increasing amount of complementary oligomer to the FAM-PNA-BC1-GO mixture, fluorescence was recovered only when FAM-PNA-BC1-1-GO complex was mixed with the complementary oligomer of BC1-1 (Fig. 4a). This was also the case with FAM-PNA-BC1-3-GO complex with the complementary oligomer of FAM-PNA-BC1-3 (Fig. 4b).
Recognition of BC1 lncRNA by the FAM-PNA-BC1-GO complex in the cells. The RT-PCR showed that mouse NE-4C neural stem cells expressed highly the BC1 lncRNAs and that human F3 neural stem cell did not, but mouse LA-4 lung adenoma cells did (Fig. 5a). Thus, NE-4C and LA-4 cells were chosen as BC1 lncRNA positive cell lines, whereas F3 cell as BC1 lncRNA negative cell line. WST-8 assay to evaluate cytotoxicity of GO yielded that the viability of NE-4C and LA-4 cells decreased gradually as GO dose increased. GO did not exhibit cytotoxicity at the concentration (4 μg/mL) used in the present study (Supplementary Figure S2). In opti-MEM media as well as in the buffer, FAM-PNA were found to be completely quenched by adding GO (Supplementary Figure S3). Real-time PCR analysis for BC1 expression was performed on cell groups treated with four independent BC1 siRNAs (siBC1-1, siBC1-2, siBC1-3 and siBC1-4) or negative control (siNC). BC1 positive NE-4C (b) and LA-4 (c) cells were used to find the optimal BC1 detection probe by confirming the knockdown of BC1 for the each designed siRNA. Data are represented as mean ± SD (n = 3). *P < 0.005 and **P < 0.001 (two-tailed Student's t test).

Discussion
Amid the rapid emerging for significance of lncRNAs as a biomarker or therapeutic target, sensing technique for lncRNAs is urgently needed. In this study, as a proof-of concept study, we introduced the PNA-formed probe of lncRNAs to enhance physiological stability for lncRNA detection in cellular level. Until recently, molecular beacon (MB) system is constructed based on the fluorescence recovery of quenched fluorescent molecule in the presence of sequence-specific target molecules 20 . Despite MB's substantially high sensitivity and specificity, nuclease degradation and temperature-sensitive non-specific fluorescence recovery limits its efficacy. MBs made by 2-OMe-modified RNA, PNA, or locked nucleic acid (LNA) can protect these targeting nucleic acids against nuclease cleavage. In this study, we adopted the PNA probe for lncRNAs to enhance the stability in the cells. Unlike black-hole quencher incompletely quenching the fluorescence of MB to detect mature microRNA during neuronal differentiation 21 , GO was reported to quench almost completely the fluorescence of MB making the cellular background minimized. In addition, GO made MB resistant to nuclease and cytotoxicity of MB-GO become lower than MB themselves 22 .
PNA-GO in one study successfully detected multiple microRNAs in living cells and even could monitor target microRNAs quantitatively 19 . MicroRNA-complementary sense sequence was used as a microRNA probe to detect the endogenous presence of mature microRNAs. In our study, the lncRNA-complementary sense sequence was not used as a probe, and instead, considering the fact that lncRNA is composed of three dimensional structures 3,4 , we needed to predict the open nucleotide frame and produce the siRNA-based probe against BC1 lncRNA.
PNA-BC1-1 was the one showing the best empirical performance to reduce the amount of intracytoplasmic BC1 lncRNA, PNA of which was neutrally charged enabling stable interaction with the surface of GO with minimal non-specific binding. GO from commercial sources whose size were variable with several orders of magnitude did not work, especially the fluorescence was not recovered by BC1 sense oligomers by any of FAM-PNA-BC1-GOs or control FAM-PNA-scr. When we used relatively uniform-sized GO collected via centrifugation method, fluorescence was recovered by the addition of matching sense oligomers in a dose dependent manner, compared to fluorescence signals in FAM-PNA-scr (Fig. 4a,b). In the cell study, this selected FAM-PNA-BC1-1-GO probe could be specifically switched on in the cytoplasmic area of BC1 positive neural stem cells at 14 h after cell transfer of BC1 lncRNA sensor, indicating that FAM-PNA-BC1-1 can now be used as sensitive probe for BC1 lncRNA presence in live cells in vitro. GO are now known to be internalized via almost all mechanism including caveolae, clathrin, macropinocytosis, or translocation [23][24][25] . We exploited the characteristics of GO internalizing across cellular plasma membrane and it worked in three different cells such as NE-4C, LA-4 and F3 cells. However, GO seems not to penetrate the nuclear membrane or pass through the nuclear pore complex, showing that GO was mostly observed to be localized in the cytoplasm [26][27][28] . As many lncRNAs participate in transcriptional regulation inside the nucleus, we need to develop the methods to deliver the FAM-PNA-GO complex to the nucleus [29][30][31] . Nuclear brain specific lncRNA such Gomafu and NEAT1 will then be studied for their action.
Detecting method of the functionally active endogenous lncRNA with its open frame which can be monitored by FAM-PNA-GO complex provides useful tool for further understanding how lncRNAs are working in intracellular milieu. This technology can now be used for the investigation of the roles of lncRNA using cellular models in vivo to understand neurodevelopmental and neurological diseases 32,33 . This method can be applied for the evaluation of the new reagents affecting the action of target lncRNAs by just examining their production (presence or absence) and RNA-binding protein-medicated open-and-closure of the open active frames (dynamic action) of the sequences of lncRNAs. This technique will be also helpful to study the sponge effect 34-36 of lncRNAs against microRNAs to examine antagonistic/synergistic action 37 between microRNA and lncRNAs during various cellular processes.

BC1 lncRNA detection in cells.
Cells were seeded on 24-well cell culture plate at 4 × 10 4 cells per well and maintained for 24 h. Each fluorescent PNA probe (100 pmol per probe) was mixed with the GO (1 μg) in a 250 μL of opti-MEM media (Invitrogen, Carlsbad, CA) for 10 min at room temperature. The media were discarded and FAM-PNA-BC1-GO complexes were treated to cells for 14 h at 37 °C. After the cells were fixed with 3.7% paraformaldehyde solution, confocal images were obtained using a LSM510 confocal laser scanning microscope (Carl Zeiss Inc., Jena, Germany).