Multiplexed photo-activation 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 ( photo-cages) tethered to the 5' untranslated region (5'-UTR) of an mRNA. Synthetic photo-cages 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 photo-cages onto the 5'-UTR. However, subsequent photo-release of the cages from the mRNA transcript triggered activation of translation with single-cell spatiotemporal resolution. To achieve sequential photo-activation of two mRNAs in the same cell, we synthesized a pair of photo-cages which can be selectively cleaved from mRNA upon photo-irradiation with different wavelengths of light. Sequential photo-activation 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.