Ribonucleoside analogs bearing terminal alkynes are useful for RNA modification applications.  The alkyne serves as a substrate for copper-catalyzed azide/alkyne cycloaddition (CuAAC or �click�) reactions allowing for further manipulation of the RNA structure and its properties.  For instance, 5-ethynyluridine (5-EU) is useful for metabolic labeling of cellular RNA for subsequent purification protocols or detection/localization experiments via CuAAC reactions with biotinylated or fluorescent azides.   While 5-ethynyluridine (5-EU) is commercially available for these applications, only recently have alkyne reagents been reported that can label cellular RNA at nucleotide positions other than uridine.  Labeling other sites allows for monitoring of nucleotide-specific events in RNA (e.g. the formation of poly A tails, etc.).  Here we show the adenosine analog 7-ethynyl-8-aza-7-deazaadenosine (7-EAA) can be used to label cellular RNA and is a superior substrate for CuAAC reactions in comparison to 5-EU.  By solving crystal structures for RNA duplexes containing these analogs, we show that, like adenosine, 7-EAA and a triazole derived from it base pair with uridine and are well accommodated within an A-form helix. 7-EAA and its triazole products are recognized in RNA template strands as adenosine by AMV reverse transcriptase.  In addition, 7-EAA in RNA is a substrate for an active site mutant of the RNA editing adenosine deaminase ADAR2.  Finally, we show CuAAC reactions with 7-EAA are sensitive to the RNA secondary structure context with single stranded sites reacting faster than duplex sites.  These studies extend our understanding of the impact of these novel nucleobase analogs and set the stage for their future use in probing RNA structure and metabolism.

Click modification of RNA at adenosine: Structure and reactivity of 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenosine in RNA

247th National Meeting (2014)

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