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In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines

RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes—“molecular machines”—used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single‐molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high‐yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2′‐azido‐modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single‐molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling.