Novel melanopsin DNA aptamers developed to regulate circadian rhythm

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Melanopsin DNA aptamers that regulate the clock hands of biological rhythms have been developed by Toyohashi University of Technology and the National Institute of Advanced Industrial Science and Technology (AIST) group.

DNA aptamers can specifically bind to biomolecules to modify their function, potentially making them ideal therapeutic oligonucleotides. We examined the DNA aptamer melanopsin (OPN4), a blue light photopigment in the retina that plays a key role in using light signals to reset the phase of circadian rhythms in the central clock.

First, 15 melanopsin DNA aptamers (Melapts) were identified after eight rounds of Cell-SELEX using cells expressing melanopsin on the cell membrane. Subsequent functional analysis of each Melapt was performed in a fibroblast cell line stably expressing both Period 2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue light stimulation. Period 2 rhythmic expression over a 24-hour period was monitored in Period 2: ELuc: Thymidine kinase (TK):OPN4 stable fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance their phase by >1.5 h, whereas seven Melapts delayed their phase by >2 h. A few Melapts induced a phase shift of approximately 2 h, even in the absence of photostimulation, probably because Melapts can only partially affect the input signaling for phase shifting. Furthermore, some Melapts-induced phase shifts in Period 1::Luc transgenic (Tg) mice were used to monitor circadian rhythms by Period 1 rhythmic expression.

These DNA aptamers could have the ability to affect melanopsin in vivoIn summary, Melapts aptamers can successfully regulate the input signal and phase shift (both phase advance and phase delay) of mammalian circadian rhythms. in vitro And in vivo.

Details:

Indirectly improving the sleep-wake cycle by manipulating melanopsin’s ability to send signals to the central clock would be socially and economically beneficial.

Melanopsin is a photoreceptor protein expressed in retinal ganglion cells that absorbs blue light with a maximum absorbance of 477 nm. Melanopsin is known to play an important role in the phase resetting of the mammalian circadian clock by blue light and the rhythmic expression of clock genes, such as Period 1.2 (By1.2). The phase of the molecular circadian clock is reset by and depends on the timing of light stimulation and transient induction of Per1 by the melanopsin photoreceptor. Recently, melanopsin antagonists acquired through chemical screening of chemical libraries mainly contribute to delaying the rhythmic phase.

In this study, we used cellular systematic evolution of ligands by exponential enrichment (Cell-SELEX) to identify DNA (single-stranded DNA; ssDNA) aptamers that induce phase shifting of circadian rhythms by melanopsin. A total of 15 types of melanopsin aptamers (Melapts 1–15) were analyzed to evaluate their ability to phase shift circadian rhythms. Per2::ELuc bioluminescent oscillations in Per2:ELuc:TK:Stable Mel cells, in which a bioluminescent reporter tracks the Per2 promoter region controlling an enhanced green-emitting luciferase from Pyrearinus termitilluminanswith overexpressed melanopsin under the control of the thymidine kinase (TK) promoter. In these stable fibroblast cell lines, the signaling pathway is incorporated into a fibroblast cell that mimics the signaling pathway from the retina to the master central clock (suprachiasmatic nucleus or nuclei: SCN) through melanopsin.

DNA aptamers are short single-stranded RNA/DNA molecules that can selectively bind to specific targets, proteins, peptides, and other molecules and can be used clinically to modify the function of target molecules. The main advantages of these aptamers are their high target specificity, lack of immunogenicity, and ease of synthesis.

Among the 15 melanopsin DNA aptamers (Melapts), four Melapts induced a phase advance and seven Melapts induced a delay of circadian rhythms (by > 1.5 h and > 2 h, respectively) in the Per2::ELuc cell line. Some Melapts induced phase shifts of approximately 2 h, even in the absence of photostimulation in vitro.

Melapt04 and Melapt10 induced a phase advance or delay of the circadian clock by approximately 3 h, respectively, at both CT22 and CT8 during the photosignal input process. This suggests that Melapt04 regulates the phase of circadian rhythms and facilitates sleep onset and wakefulness, mainly via phase advance. There are two Melaptes that advance and delay the phase shift in the same direction, regardless of the timing of the photostimulus. However, the three Melaptes advanced and delayed the phase shift in opposite directions at dawn and dusk. Therefore, these Melaptes should be useful in regulating the phases of the rhythms.

We played in vivo experiences similar to those in vitro experiments to determine whether binding of Melapt to melanopsin in the retina projecting to the SCN affected phase shifts of the central clock in the SCN. Per1::Luke transgenic mice: mice in which the Per1::Luke the recombinant gene was inserted into the genome of all cells. Per1::Luke is a recombinant gene in which the Per1 The promoter region is monitored by a firefly-derived luciferase enzyme as a reporter to monitor circadian rhythms.

Eight types of phase shift responses elicited by Melapt in Per2 expression rhythms in in vitro experiments were injected into the eye bulbs of Per1::luc Tg mice at CT22. Melapt01, Melapt03, Melapt04, Melapt07, Melapt09 and Melapt10 showed similar phase shifting abilities as Per2:ELuc:TK:Stable Mel Cells: in vivo And in vitro.

The effect of Melapt on phase shift in in vivo experiences can be predicted from in vitro experiments. Furthermore, total phase shifts were limited to 3 h in intact animals, regardless of the magnitude of Melapts’ advance or delay in Per2:Eluk:TK:Mel Cells.

In conclusion:

In summary, Melapts were able to regulate input signals and phase shifts to achieve both phase advance and phase delay in mammalian circadian rhythms. in vitro And in vivo.

Melapts could contribute to future research focused on resetting the phases of the circadian clock. Melapts could help us better adapt to modern social life cycles, enable improved crops and domesticated animals for greater productivity, and help shift workers overcome social jet lag by adjusting the phases of the circadian clock. These Melapts could help reset the phase of circadian clocks in photic input pathways.

Funding body:

This study was supported by research funds from TechnoPro Inc., TechnoPro R&D Company, and the Young Scholars Promotion Program in Advanced Interdisciplinary Research (RN). Funding for Kiban Scientists (RN 24590350 and 20H00614) was obtained from the Japan Society for the Promotion of Science (JSPS), the Mitsubishi Science Foundation (RN), and a Research Grant for Scientific and Technological Innovation at Toyohashi University of Technology (RN). This study was also supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (YN 21H02083).