Melanopsin

Melapts were amplified and labeled with a 6-FAM-forward primer. Briefly, 10 pmol Melapt, 20 pmol 6-FAM-forward primer (5′-AAAGGGGAATTCGGATCC-′3, Hokkaido System Science), 2 pmol reverse primer (5′-AAACGGAAGCTTCTGCAG-3′, Fasmac Corporation), 1 unit Go Taq DNA polymerase (Promega), 125 pmol dNTPs (Promega), 5× Go Taq Reaction Buffer (Promega), and nuclease-free water were combined in a 20 μL reaction volume using the Cell-SELEX amplification profile (Figure 2A).
Mouse fibroblast cells were cultured in 96-well plates (1.5 × 10⁵ cells/well) using 200 μL DMEM, transfected with melanopsin, and incubated at 37°C and 5% CO₂ for 24 h. Then, 6-FAM-Melapt was added to the culture medium at six different concentrations (1.405 μg/mL, 5.625 μg/mL, 11.25 μg/mL, 22.5 μg/mL, 45.0 μg/mL, and 90.0 μg/mL) and incubated for 15 min. After washing, the binding capacity of 6-FAM-Melapts was estimated by measuring the fluorescence signal in (+) melanopsin cells (λex 495 nm, λem 517 nm) using an Infinite 1,000 microplate reader (Tecan, Zürich, Switzerland; Figure 1). The most suitable concentration of Melapts was 22.5 μg/mL. Before performing each measurement, 6-FAM-Melapt was added to three different wells (n = 3). A stable mouse fibroblast cell line (Per2:ELuc:TK:Mel) with Melapts was established to monitor the phase shift of Per2:ELuc bioluminescent emission rhythms using photo-responsive ELuc.
The 5′ flanking region of Per2 (from −2,858 to +144, where +1 indicates the putative transcription start site) was PCR-amplified from the C57BL/6 J mouse genome and cloned into the XhoI and BglII sites of pELuc (PEST)-test (Toyobo, Osaka, Japan). Expression cassettes containing early poly-A (pA) signal, Per2 promoter, ELuc-PEST, and late pA signal were amplified by PCR and cloned into pENTR-D-TOPO (Thermo Fisher Scientific Inc., Waltham, MA, United States), with the attL1 and attL2 sites flanked by the upstream and downstream regions of early and late pA signals, respectively, resulting in pmPer2-ELuc-PEST-pENTR. The expression cassette was recombined into the pBsd-R4 attB vector (a gift from Dr. T. Ohbayashi) by the LR reaction using LR Clonase II Plus Enzyme Mix (Thermo Fisher Scientific, Inc.), yielding pR4-Bsd-mPer2-ELuc-PEST.
Furthermore, mOpn4-Flag, wherein the Flag-tag was fused in-frame to the C-terminus of mouse melanopsin cDNA, was synthesized as double-stranded DNA (GenScript, Tokyo, Japan) and cloned into pUC57. The mOpn4-Flag was excised using NcoI and XbaI and ligated into the NcoI and XbaI site of pTK-SLG-pENTR-D-Topo (Tabei et al., 2017 (link)), from which the SLG cDNA was removed, yielding pTK-mOpn4-Flag-pENTR. The expression cassette containing TK promoter, mOpn4-Flag, and late pA signal was recombined into the pNeo-ϕC31 attB vector (Yamaguchi et al., 2011 (link); a gift from Dr. T. Ohbayashi) by the LR reaction, resulting in pϕC31-Neo-mOpn4-Flag.
Mouse fibroblast A9 cells harboring the multi-integrase mouse artificial chromosome (MI-MAC) vector (Takiguchi et al., 2014 (link); kindly provided by Dr. M. Oshimura and Dr. Y. Kazuki) seeded into six-well plates were co-transfected a day later with pR4-Bsd-mPer2-ELuc-PEST and the R4 integrase expression plasmid pCMV-R4 (Yamaguchi et al., 2011 (link); kindly provided by Dr. T. Ohbayashi) and subcultured for selection with 6 μg/mL Blasticidin S (Thermo Fisher Scientific, Inc.). Selected cells were further co-transfected with pϕC31-Neo-mOpn4-Flag and ϕC31 integrase expression plasmid pCMV-ϕC31 (Yamaguchi et al., 2011 (link); kindly provided by Dr. T. Ohbayashi) and subcultured for selection with 800 μg/mL G418 (Nacalai Tesque, Kyoto, Japan). Genomic PCR confirmed the integration of the transgenes into the corresponding sites in the MI-MAC vector. The established cell line was named Per2:ELuc:TK:Mel.
The photo-responsive fibroblast stable cell line for functional analysis of DNA aptamers (ELuc:Per2:ELuc:TK:Mel) was stably transfected into Per2-enhanced green-emitting luciferase cells (Per2:ELuc) with melanopsin (mOPN4) expression under the control of the TK promoter to generate photo-responsive A9 fibroblast cells (Nakajima et al., 2010 (link)). Screening of DNA aptamers was performed using blue light-responsive and bioluminescence real-time imaging of circadian rhythms (Supplementary Figure S2). Per2:ELuc stably expresses ELuc under the control of the Per2 promoter because the phase due to the transcriptional activity rhythm of Per2 can be monitored from the emission rhythms of ELuc (Nakajima et al., 2010 (link)). Per2:ELuc:TK:Mel stable cells constitutively and stably express melanopsin on the cell surface under the control of the TK promoter. Melanopsin expressed on the surface of the cell membrane transmits external photo-stimuli into the cell and transiently induces Per2 transcription in the cell nucleus. Thus, Per2:ELuc:TK:Mel stable cells are suitable for the phase shift of circadian rhythms in response to blue-light photo-stimuli.

A random ssDNA library for screening of DNA aptamers by the Cell-SELEX method was designed based on a random DNA aptamer (5‘-AAAGGGGAATTCGGATCC-N-40-CTGCAGAAGCTTCCGAAAA-3′) with regions of 40 bases, and 10¹⁷ types of DNA aptamers with a fixed leading and trailing region were prepared by Hokkaido System Science (Hokkaido, Japan). A floating PC12 cell (obtained from the Cell Bank, RIKEN BRC, Ibaraki, Japan) was used to perform the Cell-SELEX method, and cells were cultured in low-glucose DMEM (FUJIFILM Wako, Tokyo, Japan) containing 10% fetal bovine serum (FBS; Wako, Tokyo, Japan), 5% donor horse serum (DHS; Wako, Tokyo, Japan), and 1% penicillin–streptomycin (Wako, Tokyo, Japan). The melanopsin expression plasmid (provided by Professor Ueda of the University of Tokyo) was transfected into PC12 cells using Lipofectamine 3000 (Invitrogen, Massachusetts, United States) to prepare (+)melanopsin-transfected PC12 cells ([+] melanopsin cells) in which melanopsin was overexpressed on the cell membrane, whereas in (−) melanopsin-free PC12-negative control cells (other [−] melanopsin cells without transfecting melanopsin: negative selection), melanopsin was not expressed. First, DNA aptamers were mixed and incubated with (−) melanopsin cells, and only unbound DNA aptamers were recovered. Second, the recovered DNA aptamers in solution were mixed and incubated with (+) melanopsin cells, and only DNA aptamer bound to cells was recovered. Then, the recovered DNA aptamers specifically bound to (+) melanopsin cells but not bound to (−) melanopsin cells were amplified by asymmetric PCR amplofication (Figure 1A).
The obtained Melapts were amplified with forward and reverse primers in a 10:1 ratio by asymmetric PCR with reactions containing 10 pmol Melapt, 20 pmol forward primer (5′-AAAGGGGAATTCGGATCC-3′, FASMAC), 2 pmol reverse primer (5′-AAACGGAAGCTTCTGCAG-3′, FASMAC), 5 units Go Taq DNA polymerase (Promega; Wisconsin, USA), 30 nmol Mg²⁺ (Promega), 2.5 nmol dNTPs (Promega), and PCR buffer (Promega) in a final volume of 20 μL. The PCR amplification profile for Melapts involved preliminary denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 4 min.
Only those amplified as a significant sense band in gel electrophoresis were used in the next round of Cell-SELEX. These steps were repeated for eight rounds to concentrate DNA aptamers by asymmetric PCR amplification. Then, DNA aptamers were cloned into a T vector (Invitrogen), and aptamer sequences were confirmed by Fasmac Corporation (Kanagawa, Japan; Table 1). Finally, 15 DNA aptamers for melanopsin were selected and named Melapt1-Melapt15 (Supplementary Figure S1). Melapts obtained by the Cell-SELEX method appeared to bind specifically to melanopsin alone. Melanopsin-KO cells and melanopsin-KO mice were not used in this study.