Capture and Analysis of Newly Synthesized RNA –A Novel Enabling Technology to Study High Resolution Gene Expression.

Newly transcribed RNA contains valuable information of the transcriptome that cannot be obtained from total RNA pool. Development of enabling technologies using affinity nucleosides will help accelerate research that depends on high resolution gene expression. We describe an enabling tool based on click chemistry, which begins with metabolic incorporation of an ethynyl uridine (EU) into newly synthesized RNA. The purified total EU‐RNA is reacted with biotin‐azide using copper catalyzed click chemistry followed by binding to streptavidin beads. The captured nascent RNA can be analyzed by RT‐qPCR, micro arrays & sequencing. Toxic effects of EU on cell health and transcriptome were investigated. Our results show that nascent RNA can be captured and analyzed via RTqPCR and RNA‐seq. No toxic effects on cell health were seen. Microarray analyses show no alteration of the global transcriptome. For the first time, this method enables pulse‐chase of RNA resulting in capture of new RNA and its identification. The main limitation of all other approaches is that true mRNA turnover cannot be studied. Using our approach, we show RNA turnover rates for several mRNA transcripts and correlation to relative abundance.