Mitochondrial Transcription: A click-chemistry derived detection methodology forgoing the use of radiation in in vitro analyses.

Abstract

Mitochondrial transcription is key for mitochondrial biogenesis, essential for both gene expression and mtDNA replication. Because of the difficulty of studying the process in vivo, studies of mitochondrial transcription have largely relied on in vitro approaches. Existing methods are based on incorporation of a radioactively labeled nucleotide to facilitate detection of the product while not perturbing the transcription reaction. However, they are difficult to use and cumbersome, preventing their widespread utilization. Here we report a new non-radioactive approach for the in vitro study of mitochondrial transcription that relies on the bio-orthogonal click chemistry reaction, utilizing click-chemistry ready azide-labeled UTP in the mitochondrial transcription system. Our approach recapitulates results obtained using radioactive methods and can be carried out using the reaction conditions typically used for in vitro radioactivity assays.