Team:Lethbridge/Project Production
Project Production
dsRNA Production:
Our strategy for dsRNA production is a multi-part approach. The construct is expressed dicistronically, with a his-tagged MS2 coat-protein expressed initially. Additionally, we employ the use of a hammerhead ribozyme fused to a theophylline aptamer, termed an aptazyme (Win, M.N., Smolke C.D. 2007). Upon addition of theophylline, the aptazyme undergoes a conformational change, resulting in cleavage at a specifically prescribed nucleotide. This theophylline aptazyme is placed just upstream of an MS2 coat-protein binding site. This allows us to purify only full-length RNA once it has been transcribed, as those transcripts not harbouring the binding domain will not be effectively purified. His-tagged MS2 coat-protein is then free to bind the newly transcribed binding domain, and using affinity chromatography, purification of the mS2 coat-protein and the bound RNA is possible. Upon addition of theophylline, cleavage and liberation of a single stranded RNA occurs, allowing for purification of a single strand of highly pure RNA.
Uleth15_purificationstrategy.jpg
By using two complementary RNA-generating sequences within the aptazyme construct, we are able to generate double stranded RNA for use in pest control simply by annealing the two resultant strands produced by our purification strategy.