Distinct populations of primary and secondary effectors during RNAi in C. elegans.
Pak, Julia and Fire, Andrew (2007) Science 315:241.

The authors identified “secondary siRNAs” (synthesized by an RNA-directed RNA polymerase) as the majority of small RNAs found during ongoing RNA interference (RNAi) in C. elegans. Terminator™ 5’-Phosphate Dependent Exonuclease was employed as one tool to characterize the 5’-termini of these siRNAs. The authors reported that the siRNAs were resistant to Terminator Exonuclease digestion indicating something other than a monophosphate at their 5’-end. Sequential treatment with alkaline phosphatase (to remove 5’-phosphates), T4 polynucleotide kinase (to add a single 5’-phosphate) and Terminator Exonuclease resulted in degradation of the siRNAs, thus providing evidence that the siRNAs have multiple phosphates at their 5’-ends. Further analysis lead the authors to conclude that these siRNAs have a 5’-triphosphate.