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.

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.

This graph shows flow cytometry data with FITC on 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. The red line, dsROX, is a positive control that acts as the product of a strand displacement reaction. The green line, no input, shows cells that were nucleofected with only the reporter, and we see that for higher levels of green fluorescence (indicating higher nucleofection efficiency), red levels stay low (indicating no strand displacement). The blue line, input, shows cells that were nucleofected with the reporter and the correct input, and we see that higher levels of green correspond to higher levels of red fluorescence (indicating strand displacement occurred).


Experimental Design: We were also able to repeat our foundational In Vivo RNA Strand Displacement results using nucleofection. This experiment was performed following the <a href="http://www.lonza.com/products-services/bio-research/transfection/nucleofector-technology.aspx">Lonza</a> protocol to nucleofect HEK293 cells. According to this protocol (but at 4C to better protect the double strand from melting), we delivered the controls and correct inputs and reporters to the cells. The concentration of the reporter was 500nM whereas the input was 1.5uM. In the above figure, it is clear that the cells that received reporter and input (blue line) have a higher red fluorescence than the one that received only the reporter (green line). This is compatible with the hypothesis that the displacement caused by the input unquenced the Rox consequently increasing the red fluorescence (dsROX is our positive control). these experiments are at early stage and further investigation is needed.

Conclusion of Results: We can also demonstrate RNA strand displacement In Vivo using nucleofection.

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.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]