Rolling circle amplification (RCA) is a method of isothermal amplification of circular DNA templates. This enzymatic process of primer extension combined with DNA strand displacement generates a long single-stranded DNA containing a repeated sequence complementary to the circular DNA template. When the long ssDNA product of RCA is decorated by hybridization to many complementary oligonucleotides labeled with a fluorescent molecule, the resulting RCA product is a single molecule with a strong signal that is labeled with multiple fluorescent molecules. The RCA reaction is currently used to enhance the signal of an analyte when detecting DNA or RNA in tissue or cell samples. DNA sequencing can be achieved directly in a section of fixed tissue or cell sample. This review describes the development of RCA-based methods for DNA and RNA detection in cells and tissues over the past decade.

Vascular endothelial growth factor (VEGF) is a secreted protein that promotes angiogenesis, and VEGF-A is an important molecule that binds to vascular endothelial growth factor receptor (VEGFR)-1 and VEGFR-2 and activates VEGF signaling pathway. We have successfully identified a set of nucleic acid aptamers that bind specifically to VEGFR-1 and VEGFR-2 using a method with systematic evolution of ligands by exponential enrichment (SELEX) and next-generation sequencer that can identify even low-frequency sequences. In this comprehensive paper, we summarize the structural analyses of the obtained nucleic acid aptamers and their functions as (1) angiogenesis promoters and (2) signaling aptamers.

In contrast to well-established DNA-selective dyes for live cell imaging, RNA-selective dyes have been less developed due to the challenge of making small molecule selectively target RNA over DNA. Two kinds of dyes (SYTO RNA select and Nucleolus Bright) are now commercially available for nucleolar RNA imaging in cells, but these two dyes are not applicable to living cells. Here we report on unsymmetrical monomethine cyanine dyes for imaging of nucleolar RNA in living cells, including green-emissive thiazole orange (TO: λ_em = 532 nm) and its regioisomer (2TO: λ_em = 532 nm), yellow-emissive benzo[c,d]indole-oxazolo[5,4-c]pyridine (BIOP: λ_em = 570 nm), and deep-red emissive benzo[c,d]indole-quinoline (BIQ: λ_em = 657 nm) and its derivative having an amino group-terminated side chain (BIQ-NH₂: λ_em = 665 nm). These cyanine-based probes were essentially non-fluorescent in the free state (Φ_free: TO, 0.00042; 2TO, 0.00009; BIOP, 0.00038; BIQ, < 0.0001; BIQ-NH₂, 0.00062), and the value of fluorescence quantum yield significantly increased in the bound state (Φ_bound: TO, 0.16; 2TO, 0.11; BIOP, 0.52; BIQ, 0.0085; BIQ-NH₂, 0.020). Among these probes, TO, 2TO and BIOP were even applicable to wash-free imaging of living cells due to their high brightness and/or remarkable light-up property. Considering their good photostability, low cytotoxicity and easy preparation as well, we expect that a series of these cyanine dyes would be a candidate for practical use toward spatiotemporal analysis of nucleolar RNA in living cells.

In this study, a photoswitching protein, named Magnet, based on protein engineering of a fungal photoreceptor was developed. Additionally, using Magnet, the author is developing optogenetic tools to manipulate the genome with blue light. The genome consists of more than 20000 genes and is essential for most biological phenomena. To understand these biological phenomena, including diseases, and to utilize or modify them, approaches that enable optical control of the genome are required. The author has developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9), the nuclease activity of which is switchable with light. PA-Cas9 allows for direct editing of the DNA sequence of the genome by light stimulation. The author has also developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR–Cas9 system. It is demonstrated that this optogenetic tool allowed for rapid and reversible targeted gene activation by light. Using this tool, optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs) was demonstrated. The CRISPR–Cas9-based photoactivatable transcription system offers a simple and versatile approach to precise gene activation. Recently, the author also developed a red light-responsive photoswitching protein, named MagRed, for deep tissue optogenetics. Genome engineering technology and optogenetics technology have emerged as different technologies. The studies described above merge these emerging research fields together.

In this study, we have developed the activation method of thermo-responsive TRPV1 ion channels of neurons using photothermal and biodegradable polymer micelles. Indocyanine Green (ICG) dye was loaded on polymer micelles formed from poly(ethylene glycol) block copolymer possessing poly(lactic-co-glycolic acid) (PEG-b-PLGA) segment. The obtained ICG-loaded micelles showed a photothermal effect under tissue penetrable near-infrared (NIR) light irradiation. In addition, the ICG micelles also displayed biodegradability in acidic conditions. Furthermore, anti-TRPV1 antibody installed ICG-micelles showed specific recognition against TRPV1 on the cell membrane and accelerated Na⁺ influx into the cells was observed under NIR irradiation. Based on these results, the ICG micelles developed in this study are expected to be used as a tool for the remote activation of neurons, and they can be applied for various manipulation and analytical techniques of the nervous system.

Epithelial-mesenchymal transition (EMT) is a dynamic cellular phenotypic change from an epithelial-like to a mesenchymal-like phenotype, which leads to functional changes in cell migration, invasion and metastasis. Recent research showed EMT is not only induced by soluble factors and gene expression but also by the biochemical and mechanical cues of the extracellular matrix (ECM). However, the effect of quantitative and dynamic changes in the ECM-driven biochemical cues on cellular phenotypes remains unclear. In this study, we developed a photoactivatable substrate that can change the cyclic Arg-Gly-Asp (cRGD) ligand density on a substrate surface based on the photocleavage reaction of the 2-nitrobenzyl compound. We observed the epithelial-to-mesenchymal morphological change in Madin-Darby canine kidney (MDCK) cells upon decreasing the cRGD density on the substrate by photoirradiation. In addition, fluorescence live imaging demonstrated membrane ruffling by the sudden cRGD density decreases, presumably due to the reorganization of the actin cytoskeleton. This photoresponsive cell scaffold is useful for the quantitative and qualitative analyses of the dynamic cellular response to ECM-driven signals, which is expected to help comprehensive understanding of EMT progression.

As the demand for fruit juices increases, there is a growing need for analytical techniques that contribute to ensuring quality and safety, as well as identifying and preventing counterfeiting. In this study, we developed a sensing system capable of precisely identifying fruit juices by combining a dansyl-modified poly-L-lysine (PLL-Dnc) with a pattern recognition algorithm. By mixing PLL-Dnc solutions dissolved in six different buffer solutions with varying pH and ionic strengths with eight different fruit juices, unique fluorescence patterns specific to each fruit juice were generated. Analyzing these fluorescence patterns with a pattern recognition algorithm successfully allowed for the high-precision identification of the fruit juices. This sensing system, which does not require expensive equipment or specialized techniques, has the potential to offer new opportunities for product development and quality assessment in the beverage industry.

Micro-intestine models have attracted attention for studies on intestinal absorption and microbiota. In order to create the intestinal model, it is necessary that monolayer-cultured intestinal epithelial model cells form tight junctions between cells, and to confirm this, measurement of trans-epithelial electrical resistance (TEER) is required. Therefore, we aimed to develop a micro-intestine model for TEER measurement that can be fabricated in a general biochemistry laboratory. We constructed a micro-intestine model by culturing Caco-2 cells as human intestinal epithelial model cells on a microdevice made of polydimethylsiloxane, polyester porous membrane, and platinum wires. The TEER value increased with time as the cells grew, indicating that the developed model is practically useful.