Team:Lethbridge HS/Contribution
Contribution
A new method for siRNA production
The method is based off the 2020 paper by Hannes Feyrer et al. in the Petzold lab at the Karolinska Institutet in Stockholm, Sweden (DOI: 10.3390/molecules25051142). As many iGEM teams can attest, production of RNA through the standard in vitro transcription by T7 RNA polymerase can be quite difficult. This new method attempts to produce RNA of precise length, improving yields and simplifying downstream purification steps.
To produce short siRNA fragments, the Petzold group designed DNA templates containing tandem repeats of the target RNA sequence. Once transcribed, the RNA could be cleaved by RNase H guided by a chimeric cleavage splint. This would result in single repeat units of the desired length.
The tandem repeats can be placed after a T7 RNA polymerase promoter. In our design we chose to use BBa_I719005 since it also contains the preferred initiation sequence for the polymerase enzyme. This construct can be placed in any plasmid for ease of storage and propagation. Following linearization of the plasmid, the DNA can be transcribed as a single long chain until further processing.
We chose to include the iGEM prefix and suffix sequences in our DNA design for ease of cloning into pSB1C3 plasmid. This also allowed us to amplify the gene using PCR to create more template for the in vitro transcription reaction.
More testing by our team and other iGEM teams is needed to determine whether this new method is more efficient than other siRNA production methods but we think this is a very positive start for the community.