Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery

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 tC O , a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tC O 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 tC O 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 tC O -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 tC O -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.

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

“…These studies and others have provided important novel insights on isolated biological systems, while there is a notable lack of FBA applications in more complex biological matrixes. In fact, the first report where an FBA was used to achieve fluorescent properties in live cells was recently published by us, where we demonstrated the applicability of the cytosine analogue tC O for studying delivery of functional mRNA to human cells 31 .…”

Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides

“…Further characterization of these granular structures will be necessary to understand their components and formulate hypothesis relating to their physiological relevance. (44), and found little effect on protein translation, underscoring the compatibility of these structures with biological systems. Beyond tracking localization and metabolism, analogous approaches could be utilized to incorporate diversely functionalized nucleoside analogs as biophysical probes to explore 16 nucleic acid structure and protein-RNA interactions in their native context.…”

“…The bicyclic and tricyclic cytidine analogs explored herein likely represent a privileged scaffold for RNA metabolic labeling, and further investigation of this class of modified nucleosides together with UCK2 engineering should lead to the development of fluorescent nucleosides with enhanced and varied spectral properties and context-dependent fluorescence. Esbjorner and Wilhelmsson recently investigated the biological properties of synthetic mRNA containing the related tC° fluorescent nucleobase analogue(44), and found little effect on protein translation, underscoring the compatibility of these structures with biological systems. Beyond tracking localization and metabolism, analogous approaches could be utilized to incorporate diversely functionalized nucleoside analogs as biophysical probes to explore nucleic acid structure and protein-RNA interactions in their native context.…”

Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics

“…This approach is however accompanied by some challenges. For example, cyanine dyes such as Cy5 and Cy7 ( Figure 3 b) can be chemically linked to RNA molecules [160] , [161] , [162] , [163] , but these bulky and hydrophobic groups can significantly interfere with translation dynamics [164] . Other dyes, such as the more hydrophilic Alexa Fluor® 647 and 750, have also been used to label siRNA [120] , [165] .…”