Part:BBa_K779501
Long reporter top strand MammoBlock
Long reporter top strand to be annealed with Part:BBa K779502.
Modifications: 5' Iowa Black RQ quencher, 3' Alexa 488 florophore, all bases modified with 2'O Methyl (ordered as a DNA oligo with Us instead of Ts).
IDT DNA oligo order: /5IAbRQ/mCmAmCmCmCmAmCmUmCmCmUmCmUmCmCmCmAmCmCmA/3AlexF488N/
Usage and Biology
When annealed to Part:BBa K779502 acts as a reporter for strand displacement reactions.
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This graph shows flow cytometry data with FITC on the x-axis in A.U. and Texas-Red on the y-axis. A 200-point moving average was taken to reduce noise in the data. We can see that for higher levels of green fluorescence (indicating transfection efficiency) in the FITC channel, the no input well and scrambled input wells have consistent low levels of red fluorescence (indicating no strand displacement). However, for the input well, higher levels of green fluorescence correlate with higher levels of red fluorescence, which indicates that for higher transfection efficiency of our reporter, we see more strand displacement taking place.
Experimental Design: This foundational experiment has finally shown that we can demonstrate RNA strand displacement in vivo after reformulating a new design for our sequences and testing multiple iterations of the design. In this experiment, each well received 150,000 HEK293 cells in supplemented DMEM. For controls, No Transfection is a well which only received cells. The No Input well was transfected with 25 pmol of our new designed longer reporter BBa_K779501 annealed to BBa_K779502 using RNAiMAX reagent. The experimental well labeled Scrambled Input, was transfected with 25 pmol of both the reporter and an input strand with the correct toehold but incorrect binding domain, which would not yield a strand displacement reaction. The experimental well labeled Input, was transfected with 25 pmol of both the reporter and the input strand with the correct toehold and binding domain, which can bind to the exposed toehold of the reporter and through branch migration, knock off the top strand of the reporter, yielding an unquenched fluorophore and successful strand displacement reaction In Vivo.
Conclusion of Results: We can demonstrate RNA strand displacement In Vivo using transient transfection of modified 2’-O-Methyl RNA reporters and input strands into mammalian cells.
Sequence and Features
Sequence is designed to be orthogonal to the HEK293 transcriptome (see Design). Design is improved on the part Part:BBa_K779121, with goals being a higher melting temperature, more orthogonal and longer domain.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| n/a | Long reporter top strand MammoBlock |