Control of Cellular Function by Reversible Photoregulation of Translation

Abstract

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.

Optochemical Control of Biological Processes in Cells and Animals

“…Ogasawara demonstrated reversible optical control of translation in cells by installing a styryl-modified 2′-deoxyguanosine residue as the 5′-cap of mRNA encoding a constitutively active H-Ras gene. [212] In the trans form, the styryl group inhibits translation by blocking the interaction between translation initiation factor elF4E and the 5′-cap. Delivery of the styryl-capped H-Ras mRNA into rat brain cells led to expansion and contraction of cells upon irradiation with 405 nm and 310 nm light, respectively.…”

“…[211] However,t hese technologies have not yet been applied in vivo and achieving reversible control of oligonucleotide function in living organisms remains ac hallenge.O gasawara demonstrated reversible optical control of translation in cells by installing as tyrylmodified 2'-deoxyguanosine residue as the 5'-cap of mRNA encoding ac onstitutively active H-Ras gene. [212] In the trans form, the styryl group inhibits translation by blocking the interaction between translation initiation factor elF4E and the 5'-cap.D elivery of the styryl-capped H-Ras mRNAi nto rat brain cells led to expansion and contraction of cells upon irradiation with 405 nm and 310 nm light, respectively.O ne major limitation of this tool is the requirement for potentially damaging UV-B light for photoswitching. More recently,2phenylazo groups were incorporated at the C2 amine of aguanosine residue and subsequently installed as the 5'-cap of am RNAs equence through in vitro transcription, thereby allowing photoisomerization at longer wavelengths.…”

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

“…[5] In the case of guanosine, other positions like N1, C8, or the exocyclic N 2 position have been explored albeit to am uch lower extent. [6] Forc hemoenzymatic approaches,where photocaged nucleoside triphosphates (NTPs) are synthesized and enzymatically introduced into DNAo rR NA by polymerases,t he 5p osition of pyrimidines is particularly favorable. [7] Recently,p ost-enzymatic photocaging of DNAw as also achieved by using methyltransferase (MTase)-catalyzed transfer of PC groups to the N 6 position of adenosines.…”

Synthesis and Properties of 2'-Deoxyuridine Analogues Bearing Various Azobenzene Derivatives at the C5 Position

“…Owing to their dynamic structural changes upon photo-irradiation, cis-trans type photochromic derivatives could greatly influenced the stability of nucleic acid duplexes. Although stilbene-type derivative-modified ONs have been utilized for photo-control of duplex formation, G-quadruplex formation, and gene expression, they require short wavelength photo-irradiation for cis to trans isomerization [9][10][11]. This would be disadvantageous for biomolecules due to photo-damaging reactions including the formation of pyrimidine dimers [12].…”