Light-Activated Control of Translation by Enzymatic Covalent mRNA Labeling

Abstract

Activation of cellular protein expression upon visible-light photocleavage of small-molecule caging groups covalently attached to the 5' untranslated region (5' UTR) of an mRNA was achieved. These photocleavable caging groups are conjugated to in vitro transcribed mRNA (IVT-mRNA) through RNA transglycosylation, an enzymatic process in which a bacterial tRNA guanine transglycosylase (TGT) exchanges a guanine nucleobase in a specific 17-nucleotide motif (Tag) for synthetic pre-queuosine₁ (preQ₁ ) derivatives. The caging groups severely reduce mRNA translation efficiency when strategically placed in the 5' UTR. Using this method, we demonstrate the successful spatiotemporal photoregulation of gene expression with single-cell precision. Our method can be applied to therapeutically relevant chemically modified mRNA (mod-mRNA) transcripts. This strategy provides a modular and efficient approach for developing synthetic gene regulatory circuits, biotechnological applications, and therapeutic discovery.

Keywords: RNA labeling; RNA modification; gene expression; optogenetics.

Figure 1.

RNA transglycosylation at guanosine (RNA-TAG). RNA-TAG utilizes a bacterial tRNA guanine transglycosylase (TGT) to exchange a guanine nucleobase on a specific 17-nucleotide motif (Tag) for synthetic preQ₁ derivatives. In this work, synthetic photocleavable caging groups were covalently incorporated onto the mRNA transcript.

Figure 2.

mRNA translation activity with different numbers of photocleavable caging groups incorporated. A) Cell images showing cells transfected with different mRNA constructs. Scale bar: 50 μm. B) Background-subtracted average cell fluorescence intensity, which indicates the protein expression level. Average fluorescence intensity values were measured from more than 80 cells. Error bars show the SEM.

Figure 3.

Immunoblot analysis of EGFP expression. Cells were transfected with either unlabeled RNA-Tag0, unlabeled RNA-Tag1/2/3, labeled RNA-Tag1/2/3, or labeled RNA-Tag1/2/3 that was uncaged through UV irradiation prior to transfection.

Figure 4.

Live-cell uncaging of photocaged RNA-Tag1/2/3, A) Experimental procedure for live-cell uncaging. B) Live-cell uncaging using 405 nm laser light. Red circles indicate cells transfected with photocaged RNA-Tag1/2/3 and irradiated with 405 nm laser light for 10 seconds. Scale bar: 40 μm.

Scheme 1.

Route for synthesis of the TGT substrate biotin-Bac-preQ₁ (7). Boc = tert-butyloxycarbonyl protecting group, DMF = dimethylformamide, DIEA = N,N-Diisopropylethylamine, DCM = dichloromethane, TFA = trifluoroacetic acid.