Multiplexed Photoactivation of mRNA with Single-Cell Resolution

Abstract

We demonstrate sequential optical activation of two types of mRNAs in the same mammalian cell through the sequential photocleavage of small molecule caging groups ("photocages") tethered to the 5′-untranslated region (5′-UTR) of mRNAs. Synthetic photocages were conjugated onto target mRNA using RNA-TAG, an enzymatic site-specific RNA modification technique. Translation of mRNA was severely reduced upon conjugation of the photocages onto the 5′-UTR. However, subsequent photorelease of the cages from the mRNA transcript triggered activation of translation with single-cell spatiotemporal resolution. To achieve sequential photoactivation of two mRNAs in the same cell, we synthesized a pair of photocages that can be selectively cleaved from mRNA upon photoirradiation with different wavelengths of light. Sequential photoactivation of two mRNAs enabled precise optical control of translation of two unique transcripts. We believe that this modular approach to precisely and rapidly control gene expression will serve as a powerful tool in future biological studies that require controlling translation of multiple transcripts with high spatiotemporal resolution.

Visible-to-NIR-Light Activated Release: From Small Molecules to Nanomaterials

“…Derivative 30d was shown to have similar spectroscopic and photochemical properties to 30c ( Table 7 ) 354 while providing an additional derivatization point for further modulation of its properties and functions. 354 − 360 The alkyl substituents of the (7-dialkylaminocoumaryl)methyl group can easily be replaced with other functional moieties without significantly affecting the molecule’s photophysical and photochemical properties, 361 allowing other properties to be tuned to expand the PPG’s utility. For example, long alkyl chains have been appended to the 7-amino group to increase hydrophobicity, 362 − 366 and highly polar or charged moieties such as bis(carboxymethyl), 283 , 306 , 367 − 375 bis((dimethylamino)ethyl)carboxamide, 376 and bis(ethylsulfonate) 377 , 378 groups have been used to increase water solubility and control cellular permeability.…”

Controlling gene expression with light: a multidisciplinary endeavour

“…A photocage was also installed on a nucleobase of a DNA template by using a methyltransferase with a 2-nitrobenzyl-modified S-adenosyl methionine substrate, which was used to control cell-free expression [ 40 ]. Enzymatic insertion of photocleavable biotin-coumarin-modified nucleobases into mRNA also enabled controlled expression in mammalian cells [ 41 , 42 ].…”

RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

“…RNA-TAG utilizes a bacterial tRNA guanine transglycosylase (TGT) to exchange a guanine nucleobase within a specific 17-nucleotide RNA stem loop structure (Tag) with a modified analog of the natural substrate preqeuosine1 (preQ1). Covalent modification is site-specific, robust, irreversible, and versatile, and RNA-TAG technology has been adapted to image cellular RNA in fixed cells, regulate mRNA translation, and study RNA-protein interactions [18][19][20][21][22][23]. We were curious whether or not RNA-TAG could be utilized to cyclize RNA targets with synthetic linkers.…”

“…The copyright holder for this preprint (which this version posted April 23, 2021. ; https://doi.org/10.1101/2021.04. 22.441030 doi: bioRxiv preprint 2b]. Crosslinking of RNA enables rapid identification of potential regions of inter-and intramolecular interactions as well as study of the three-dimensional structure of complex RNA [2c-2e].…”