Part:BBa_K3714010

Improved gard-337 for lateral flow assay
This is an improved version of BBa_K3714002, which is an aptazyme designed for detecting gardiquimod by lateral flow assay (LFA). We aimed to control the cleavage of aptazyme so we can transcribe them in advance to shorten overall time consumption.
To test our optimized design with TMSD(toehold mediated strand displacement) reaction, a toehold site between biotin probe binding site and aptazyme domain is necessary (Figure.1). However, designing the toehold and probe manually and testing with available RNA structure prediction model is a huge work with consuming much labor.

Figure.1 | Design of temporarily inhibition and disinhibition by oligonucleotides.

The toehold site in part was designed by our probe design model and verified by the prediction model of team: SJTU-Software. And the changed sequences shows no influence on the function of aptazyme(Figure.2, also see the unchanged BBa_K3714002), the toehold worked as our expect as well (Figure.2).
The stop oligo significantly increased the percentage of the uncleaved band, resembling the effect of gardiquimod (the inhibitor of this aptamer). Thus our engineering is successful.

Figure.2 | Verification of temporarily inhibition and disinhibition by oligonucleotides. The aptazyme was produced by a T7 in vitro transcription system. The product was run on a denatured PAGE. Gardiquimod is an inhibitor of the cleavage activity and "-gardiquimod" is a control group. "stop" and "start" are two oligonucleotides designed to control the activity of the aptazyme.

Sequence and Features

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]