Fig. 2 Cell-SELEX method for screening Melapts. Melapts that specifically bind melanopsin were selected by Cell-SELEX because melanopsin is a membrane protein bound to and accessible from outside cells (IMAGE)
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(A) DNA aptamers were selected using the Cell-SELEX method from a random ssDNA library. DNA aptamers were mixed and bound to (−) melanopsin cells (negative selection). When unbound DNA aptamers were retrieved, they were combined with (+) melanopsin cells to promote binding to melanopsin protein (positive selection). The Cell-SELEX process was performed over eight rounds, and 15 types of Melapt were screened.
(B) Conceptual diagram of Melapt binding to melanopsin followed by induction of the expression of the clock gene Per2. When melanopsin expressed on the surface of the cell membrane is photostimulated by a Melapt, the concentration of Na+ and Ca+ in the cytoplasm increases transiently, inducing the activation of transcriptional factors and the upregulation of Per2 in the cell nucleus.
(C) Phase shift of luciferase emission rhythms in Per2:ELuc:TK:Mel cells stably expressing melanopsin. When Per2:ELuc cells expressing melanopsin were photostimulated post-addition of Melapts, Per2:ELuc emission rhythms showed phase advance (green curve) or phase delay (blue curve) relative to controls (purple curve); these shifts were Melapt-dependent. PBS buffer alone was added to the control cells.
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