Part:BBa_K5086017

wt

wt (BBa_K5086017) is a single-stranded DNA sequence with 23 nucleotides. hsa-mir-99b_DDSD, hsa-mir-200c_DDSD, hsa-mir-21_DDSD, and hsa-mir-141_DDSD probes have partially complementary sequences to it at positions following the reporter probe (5' to 3'). wt strand 18nt starting at the 5' end of the strand pairs with the reporter probe and the latter 5nt does not pair with it. ' to 3'). 18nt starting at the 5' end of the wt strand pairs with the reporter probe, and the latter 5nt does not pair with the reporter probe. the presence of this free 5nt sequence prevents DNA polymerase from binding to the reporter probe between the wt strands and initiating a polymerase-mediated strand displacement reaction.
Reporting format:

Reporting format I:Its complementary sequence is also present on Universalness_Q and is longer than Universalness_F. When the wt strand is not displaced, the reporter probe is quenched due to the formation of a stable double-stranded structure that draws the fluorescent molecules closer to the quenching molecules, resulting in fluorescence resonance energy transfer (FRET) occurring; when the wt strand is displaced, the wt strand binds to the double-stranded probe through a toehold-mediated strand When the wt strand is replaced, the wt strand hybridizes with the long DNA strand in the double-stranded probe through a toe-mediated chain displacement reaction, resulting in the formation of a thermodynamically more stable double-stranded structure, while competing with the short DNA strand for fluorescence recovery, releasing fluorescence signals.

Reporting format II:When the wt strand is not replaced, the distance between the fluorescent molecule and the quenching molecule is closer due to the restriction of the DNA strand, resulting in fluorescence resonance energy transfer (FRET), and fluorescence quenching occurs; when the wt strand is replaced, it activates the paracrine activity of cas12A enzyme in conjunction with Universalness_crRNA to perform a nonspecific cleavage and cut the strand into two segments, realizing the recovery of fluorescence, releasing the fluorescence signal.
We first validated the system by electrophoresis：

Fig.First report form electrophoresis results

Fig.Second report form electrophoresis results

As shown in the figure, this result indicates that the reaction is following the process we designed.

Fig.First report form fluorescence graph

Fig.Second report form fluorescence graph

We set up a concentration gradient of the target in the solution to verify whether the system can produce a quantitative response to the target. The results of the fluorescence curves are shown in the figure, and both reporting forms can produce a quantitative response.

Fig.0 μM target concentration single molecule fluorescence analysis plot

Fig.6.25 μM target concentration single molecule fluorescence analysis plot

Fig.25 μM target concentration single molecule fluorescence analysis plot

Fig.50 μM target concentration single molecule fluorescence analysis plot

Fig.100 μM target concentration single molecule fluorescence analysis plot

At the same time, in order to achieve greater diagnostic accuracy, we will ultimately use total internal refractive fluorescence microscopy (TIRFM) for smFRET measurements and fluorescence molecular imaging with our self-developed automated analysis program to analyze multiple data points. Therefore, we also validated the method using a target concentration gradient and the results are shown in the figure, which also produces a quantitative response. Sequence and Features BBa_K5086017 SequenceAndFeatures