Melanopsin DNA aptamers can regulate input signals of mammalian circadian rhythms by altering the phase of the molecular clock.

DNA aptamers can bind specifically to biomolecules to modify their function, potentially making them ideal oligonucleotide therapeutics. Herein, we screened for DNA aptamer of melanopsin (OPN4), a blue-light photopigment in the retina, which plays a key role using light signals to reset the phase of circadian rhythms in the central clock. Firstly, 15 DNA aptamers of melanopsin (Melapts) were identified following 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 Period2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue-light stimulation. Period2 rhythmic expression over a 24-h period was monitored in Period2:ELuc stable cell line fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance phase by >1.5 h, while seven Melapts delayed phase by >2 h. Some Melapts caused a phase shift of approximately 2 h, even in the absence of photostimulation, presumably because Melapts can only partially affect input signaling for phase shift. Additionally, some Melaps were able to induce phase shifts in Per1::luc transgenic (Tg) mice, suggesting that these DNA aptamers may have the capacity to affect melanopsin in vivo. In summary, Melapts can successfully regulate the input signal and shifting phase (both phase advance and phase delay) of mammalian circadian rhythms in vitro and in vivo.

Restricted Feeding Resets the Peripheral Clocks of the Digestive System.

All organisms maintain an internal clock that matches the Earth's rotation over a period of 24 h, known as the circadian rhythm. Previously, we established Period1 luciferase (Per1::luc) transgenic (Tg) mice in order to monitor the expression rhythms of the Per1 clock gene in each tissue in real time using a bioluminescent reporter. The Per1 gene is a known key molecular regulator of the mammalian clock system in the autonomous central clock in the suprachiasmatic nucleus (SCN), and the peripheral tissues. Per1::luc Tg mice were used as a biosensing system of circadian rhythms. They were maintained by being fed ad lib (FF) and subsequently subjected to 4 hour (4 h) restricted feeding (RF) during the rest period under light conditions in order to examine whether the peripheral clocks of different parts in the digestive tract could be entrained. The peak points of the bioluminescent rhythms in the Per1::luc Tg mouse tissue samples were analyzed via cosine fitting. The bioluminescent rhythms of the cultured peripheral tissues of the esophagus and the jejunum exhibited phase shift from 5 to 11 h during RF, whereas those of the SCN tissue remained unchanged for 7 days during RF. We examined whether RF for 4 h during the rest period in light conditions could reset the activity rhythms, the central clock in the SCN, and the peripheral clock in the different points in the gastrointestinal tract. The fasting signals during RF did not entrain the SCN, but they did entrain each peripheral clock of the digestive system, the esophagus, and the jejunum. During RF for 7 days, the peak time of the esophagus tended to return to that of the FF control, unlike that of the jejunum; hence, the esophagus was regulated more strongly under the control of the cultured SCN compared to the jejunum. Thus, the peripheral clocks of the digestive system can entrain their molecular clock rhythms via RF-induced fasting signals in each degree, independently from the SCN.

Nanoscale-tipped wire array injections transfer DNA directly into brain cells ex vivo and in vivo.

Genetic modification to restore cell functions in the brain can be performed through the delivery of biomolecules in a minimally invasive manner into live neuronal cells within brain tissues. However, conventional nanoscale needles are too short (lengths of ~10 µm) to target neuronal cells in ~1-mm-thick brain tissues because the neuronal cells are located deep within the tissue. Here, we report the use of nanoscale-tipped wire (NTW) arrays with diameters < 100 nm and wire lengths of ~200 µm to address biomolecule delivery issues. The NTW arrays were manufactured by growth of silicon microwire arrays and nanotip formation. This technique uses pinpoint, multiple-cell DNA injections in deep areas of brain tissues, enabling target cells to be marked by fluorescent protein (FP) expression vectors. This technique has potential for use for electrophysiological recordings and biological transfection into neuronal cells. Herein, simply pressing an NTW array delivers and expresses plasmid DNA in multiple-cultured cells and multiple-neuronal cells within a brain slice with reduced cell damage. Additionally, DNA transfection is demonstrated using brain cells ex vivo and in vivo. Moreover, knockdown of a critical clock gene after injecting a short hairpin RNA (shRNA) and a genome-editing vector demonstrates the potential to genetically alter the function of living brain cells, for example, pacemaker cells of the mammalian circadian rhythms. Overall, our NTW array injection technique enables genetic and functional modification of living cells in deep brain tissue areas, both ex vivo and in vivo.

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor.

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 103 cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.