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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.

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Hybridization of nucleic acids through Watson-Crick base pairing is a fundamental phenomenon in many biological events, such as gene regulation [1] by antisense agents, [2] small interfering RNA (siRNA), [3] or microRNA (miRNA). [4] In addition, the ability to form duplexes and other secondary structures through predictable hybridization has been used in constructing programmable devices and architectures on the nanometer-length scale.[5] Therefore, the necessity for methods that control hybridization by external stimuli is clear. The most promising external trigger is photoirradiation because it allows accurate and easy control of the location, dosage, and time when an event occurs. One common approach for photoregulation of hybridization involves the installation of a photoprotecting group that can be completely removed by photoirradiation.[6] This strategy, termed caging, [7] allows regulation only once and in only one direction, whereas the method employing cis-trans photoisomerization of azobenzene inserted as a base-pair (bp) replacement allows reversible control.[8] Although a significant melting-temperature difference (DT m ) is obtained upon cis-trans isomerization of azobenzene, this approach requires the introduction of multiple azobenzene moieties in the side chain. For example, 9 azobenzenes are required for the photoregulation of a 20-bp DNA duplex; these cause the structure of the duplex to deviate far from the B form and prevent its interaction with proteins. [9] Additional efforts are, therefore, needed to create more broadly applicable photoregulation methods that promise straightforward and reversible control without harm to the native B-form structure.Herein, we report a new strategy for the photoregulation of hybridization by using cis-trans photoisomerization of a photochromic nucleoside (PCN) that reversibly changes its photochemical and physical properties, such as fluorescence intensity, upon photoisomerization by an external light stimulus.[10] Our strategy successfully allowed extremely straightforward and reversible duplex regulation of a 20-bp DNA, even at room temperature. We designed three C8-substituted 2'-deoxyguanosine PCNs, G, so the stability of the duplex should change due to alteration in the steric hindrance to the backbone upon cis-trans isomerization (Scheme 1). The synthesis of the PCNs was achieved by employing two consecutive palladium-catalyzed crosscoupling reactions, as shown in Scheme 2. 8-Bromo-2'-deoxyguanosine (1) was converted into 2 by stepwise protections of the amino group with DMF-dimethylacetal and of the 5'-hydroxy group with 4,4'-dimethoxytritylchloride. Compound 2 was then subjected to cross-coupling with tributyl-

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.