Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Tom Baladi,Marcel R Hollenstein,Ivo Sarac,Anders Dahlén,Cécile Gasse,Elin K Esbjörner,Fabienne Levi-Acobas,L Marcus Wilhelmsson,Audrey Gallud,Emanuele Celauro,Jesper R Nilsson

doi:10.1021/jacs.1c00014

Abstract

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Highlights

RNA is a key molecule of life and a main active player of the central dogma of molecular biology
We find that chemically transfected tCO-labeled RNA, unlike a stateof-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2
In this study we demonstrate that tCO, a fluorescent cytosine base analogue with moderate chemical modification, in a remarkable way takes the role of natural cytosine in a number of biochemical processes

Summary

Introduction

RNA is a key molecule of life and a main active player of the central dogma of molecular biology. Recent advances in RNA imaging have generated a broad spectrum of tools and probes by which RNA can be analyzed and quantified, but they generally rely on the use of heavily modified externally labeled oligonucleotides with chemical and physical properties that are significantly different from their natural counterparts Cyanine dyes such as Cy3 and Cy5 conjugated, for instance, via strain-promoted cycloaddition linkers remain the most common labeling choice,[6,7] even though their bulkiness and hydrophobicity significantly impede both transcription and translation[8] (has been noted by TriLink Biotechnologies, one of the main manufacturers of externally labeled mRNA) of mRNA. Development of more universally applicable RNA labeling schemes and probes that are minimally perturbing to RNA’s functions and compatible with live-cell fluorescence imaging is needed and may become as crucial for the RNA field as the discovery and Received: January 1, 2021 Published: April 2, 2021

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