Shinzi Ogasawara

Reversible Photoswitching of a G-Quadruplex

G-quadruplexes are formed by stacked G-quartets, a planar association of four guanines, by Hoogsteen hydrogen bonding. They are found in guanine-rich sequences and are involved in several key biological events. For example, telomeric DNA consists of long (TTAGGG)n repeats, and can fold into an intramolecular G-quadruplex that is required to stabilize the ends of chromosomes for proper replication and segregation of eukaryotic chromosomes. This G-quadruplex can also inhibit telomere elongation by telomerase, which is expressed in 85–90 % of tumor cells. Furthermore, Gquadruplexes in the promoter regions of c-myc, RET, KRAS, c-kit, VEGA, HIF-1a, and bcl-2 or in the 5’UTRs of Zic-1 and NRAS mRNA may modulate transcription or translation. G-quadruplexes are also sometimes found in aptamers, which are small, structured single-stranded nucleic acids selected from vast combinatorial libraries using the SELEX method. They bind with high affinity and specificity to their target molecules and prevent their activity. For example, (GGGT)4 or (GGGC)4 can form Gquadruplexes in the presence of potassium ions, and can protect host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) by binding to integrase. Similarly, anti-thrombin aptamers form intramolecular G-quadruplexes that bind to and inhibit thrombin, which plays an important role in thrombosis and hemostasis. The possibility of regulating biological processes by controlling G-quadruplex formation with external stimuli is both exciting and challenging. The most promising external trigger is light, as it allows accurate and easy control of the location, dosage, and timing of stimulation. Heckel and co-workers have developed a light-controllable anti-thrombin aptamer with a photoprotecting group that can be completely removed by photoirradiation. Unfortunately, however, such uncaging methods are irreversible. We recently developed a new strategy for photoregulating nucleic acid structures using cis–trans photoisomerization of a photochromic nucleobase (PCN), which reversibly changes its photochemical and physical properties upon exposure to external light stimuli. This method permits efficient, reversible duplex regulation even at room temperature. Herein, we describe a novel method of photoregulating G quadruplexes by cis–trans photoisomerization of 8fluorenylvinyl-2’-deoxyguanosine (G). G can be photoisomerized highly efficiently from trans to cis by irradiation at 410 nm, and back to trans at 310 nm (Scheme 1). To show the potential applications of our method, we performed reversible photoregulation of the ability of G-quadruplex aptamers to bind with thrombin (Figure 1a).

Autonomous DNA Computing Machine Based on Photochemical Gate Transition

We report the construction of a one-pot autonomous DNA computing machine based on photochemical gate transition (photocleavage, hybridization, and photoligation), and we performed binary digit additions using this machine. In our method, both photochemical DNA manipulations previously reported, photoligation via 5-carboxyvinyldeoxyuridene (cvU) containing ODN and photocleavage via carbazole-modified ODN, were employed. The binary digit additions were autonomously carried out by one-time irradiation at 366 nm in the single test tube. The fluorescence readout by the DNA chip was in good agreement with the correct answer of binary digit additions. We believe that this system is easily applicable to correlation analysis between SNPs as well as other binary digit processing, such as subtraction.

Control of Cellular Function by Reversible Photoregulation of Translation

The use of light as an external stimulus offers the potential for spatiotemporal control and is thus ideal for controlling gene expression in living cells. In commonly used caging systems, once the caging compound is removed, protein expression cannot be stopped, due to the irreversibility of the uncaging reaction. We have developed a reversible method for regulating protein expression with the aid of a photoresponsive cap that can control the translation of mRNA in a reversible manner through triggering of cis–trans photoisomerization of the cap. In its trans form, the photoresponsive cap completely inhibited translation, whereas the cis form yielded protein (12.7 times more translated protein than in the trans form). Moreover, we succeeded in controlling the levels, timing and duration of protein expression in living mammalian cells. Additionally, neuronal differentiation of PC12 cells was photoinduced by controlling constitutively active H‐Ras 61L protein expression.

Duration Control of Protein Expression in Vivo by Light-Mediated Reversible Activation of Translation

The photocontrol of protein expression enables the spatiotemporal induction of biological events in living cells or organisms. However, commonly used method such as photocontrollable transcription factor or caged nucleic acids is unsuitable for precise control of the duration of protein expression. Here, I report a photocontrollable cap (PC-cap) that can control the translation of mRNA in a reversible manner via its cis-trans photoisomerization through illumination with 370 and 430 nm light. 2-meta-Methyl-phenylazo cap (mMe-2PA-cap) in the trans form silences translation in zebrafish embryo, whereas treatment with the cis form provided a 7.1 times larger amount of translated protein compared to the trans form. Moreover, translation activated by illumination of the embryo with 370 nm light was rapidly inactivated again by subsequent illumination with 430 nm light. An application of this approach was demonstrated by photoinducing the development of double-headed zebrafish by controlling the expression period of squint protein.

Template directed reversible photochemical ligation of oligodeoxynucleotides.

We demonstrated that 5-vinyldeoxyuridine (VU) and 5-carboxyvinyldeoxyuridine (CVU) can be used to photoligate a longer oligonucleotide (ODN) from smaller ODNs on a template. By performing irradiation at 366 nm, these artificial nucleotides make photoligated ODNs with high efficiency without any side reactions. Moreover, by performing irradiation at 312 nm, these photoligated ODNs were reversed to the original ODN. VU needs to be irradiated 366 nm for 6 h, but CVU needs to be irradiated at 366 nm for 15 min. Finally, we made a self-assembled structure with an ODN containing CVU and observed the photoligated ODN by photoirradiation.

Photochromic nucleobase: reversible photoisomerization, photochemical properties and photoregulation of hybridization.

We have developed a set of photochromic nucleobases (PCNs), which reversibly change the photochemical and physical properties such as fluorescence intensity upon cis-trans photoisomerization by the external light stimuli. PCNs showed very rapid and efficient reversible photoisomerization by illumination at specific wavelength. In addition, photoisomerization can be iteratively performed by alternate illumination with 250 approximately 350 nm and 350 approximately 500 nm light without any side reactions. We demonstrated a new type of the photoregulation of hybridization using this switching property.

Photo-controllable aptamer

We have successfully developed a new method for photoregulation of G-quadruplex formation using cis-trans photoisomerization of the photochromic nucleobase (8FV)G. Our photo-controllable quadruplexes can be switched between a very stable quadruplex state and a non-structured state in a straightforward and reversible fashion. We also demonstrated reversibly control binding of a G-quadruplex aptamer to thrombin.

Reversible Photoregulation of Gene Expression and Translation

Several methods for controlling gene expression by light illumination have been reported. Most of these methods control transcription by regulating the interaction between DNA and transcription factors. The use of a photolabile protecting compound (cage compound) is another promising approach for controlling gene expression, although typically in an irreversible manner. We here describe a new approach for reversibly controlling translation using a photoresponsive 8-styryl cap (8ST-cap) that can be reversibly isomerized by illumination with light of a specific wavelength.