Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2′- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.
Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that ‘EC-tagging’ occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.
Profiling RNA expression in a cell-specific manner continues to be a grand challenge in biochemical research. Bioorthogonal nucleosides can be utilized to track RNA expression; however, these methods currently have limitations due to background and incorporation of analogs into undesired cells. Herein, we design and demonstrate that uracil phosphoribosyltransferase can be engineered to match 5-vinyluracil for cell-specific metabolic labeling of RNA with exceptional specificity and stringency.
We report a strategy for the orthogonal conjugation of the vinyl nucleosides, 5-vinyluridine (5-VU) and 2-vinyladenosine (2-VA), via selective reactivity with maleimide and tris(2-carboxyethyl)phosphine (TCEP), respectively. The orthogonality was investigated using density functional theory (DFT) and confirmed by reactions with vinyl nucleosides. Further, these chemistries were used to modify RNA for fluorescent cell imaging. These reactions allow for the expanded use of RNA metabolic labeling to study nascent RNA expression within different RNA populations.
Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.
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