Poly(A) tails, a long chain of adenosine nucleotides of variable length, are posttranscriptionally added to the 3'-end of eukaryotic mRNAs by poly(A) polymerase (polyadenylation). This modification plays a critical role in mRNA metabolism and thus gene regulation e.g. by influencing mRNA stability, nuclear export and translation efficiency. Traditional methods for the analysis of poly(A) tails do not allow the discrimination between preexisting and newly polyadenylated mRNA transcripts e.g. induced by changes in signaling pathways.
Two CLICK-functionalized adenosine analogs - N6-Propargyl-Adenosine (N6pA) and 2-Ethynyl-Adenosine (2-EA) have been reported as suitable substrates for poly(A) tail synthesis (polyadenylation) monitoring in mammalian cells. N6pA and 2-EA are cell permeable and incorporate instead of their natural analog adenosine both transcriptionally by RNA polymerase I, II and III and post-transcriptionally by poly(A) polymerase (Figure 1A). In vitro polyadenylation can be performed using the ATP analogs N6-Propargyl-ATP (N6pATP) or 2-Ethynyl-ATP (2-EATP) that serve as substrates for poly(A) polymerase from cell extracts (Figure 1B).
Figure 1: A) Principle of in vivo polyadenylation with the cell-permeable ethynyl-functionalized analogs 2-EA or N6pA, respectively. B) Principle of in vitro polyadenylation with ethynyl-functionalized ATP analogs 2-EATP or N6pATP, respectively. The resulting ethynyl-functionalized RNA (population) can subsequently be labeled via Cu(I)-catalyzed click reactions, allowing introduction of a Biotin group for subsequent purification tasks (via Azides of Biotin) or a fluorescent group for subsequent microscopic imaging (via fluorescent Azides).
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