Part:BBa_K4778008
probe-β
This part is the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR).
Usage and Biology
Sequence：5'-AGACACTGCCAUAAUUUCUACUAAGUGUAGAUGAUCGUUACGCUAACUAUGAACAACCAGCTA-3'
Length：63nt
Fig1. The structure of probe-β_LP. The blue is ribonucleic acid, the yellow is deoxyribonucleic acid.
This component is the second generation of miR-34a probe we designed. We adjust the sgRNA sequence in the probe and change the length of the sgRNA (blue) to 41nt. However, the result is still not ideal, and we think that the stability of the linear probe may be poor, so we cyclized the probe.（Fig2）。
This circularized DNA-RNA chimeric probe is one of our favorite components. This part can not only be paired with specific miRs to recognize miRs. Also because its sequence contains a complete sgRNA sequence, it can also serve as a "warehouse" for sgRNA, storing sgRNA that is unfolded into an active structure. In the second step of the reaction, when Cas12a is activated by the sgRNA from the first step, it will have trans-cleaving activity, cleave the DNA sequence of the circularized probe, and release new sgRNA folded into the correct structure. The new sgRNA will activate the new Cas12a, thereby achieving exponential amplification of the signal. We tested it using this probe and the results were very good. This simple probe assumes two functions, which not only improves the stability of the system, but also greatly simplifies our system.
Results
We have tested this probe and the results are very satisfactory. The urea denaturing PAGE showed thatThe experimental group produced distinct sgRNA bands (Fig3). We also compared the linear probe with the circular probe. Under the same reaction conditions, after the mir-linear probe complex was cleaved by DSN, the amount of remaining linear probes was reduced, but no obvious sgRNA bands were observed. After the Mir-annular probe complex was cleaved by DSN, an obvious sgRNA band were generated. This indicates that the circular probe is more stable and DSN has a higher cleavage efficiency for the mir-circular probe complex.
Fig3. Denaturing PAGE. Arrow denotes sgRNA
We monitor the final fluorescence signal generated by the system.It can be seen from the diagram that the system can correctly reflect the correct relationship of different input concentrations of miRNA, and has a better improvement than the previous three iterations, which indirectly showed that circular probe improves the work efficiency of DSN. At the same time, we found that there is still a large gap in the curves between 10E-2nM miRNA and 0Nm miRNA, indicating that under our laboratory conditions, the theoretical threshold of this system is lower than that of 10E-2nM, which is an exciting progress compared with the initial threshold of 10E-1nM.(Fig4)
Reference
1.Lu, N. Z., & Cidlowski, J. A. (2005). Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Molecular Cell, 18(3), 331–342. https://doi.org/10.1016/j.molcel.2005.03.025
2.Monsalve, G. C., Yamamoto, K. R., & Ward, J. D. (2019). A New Tool for Inducible Gene Expression in Caenorhabditis elegans. Genetics, 211(2), 419–430. https://doi.org/10.1534/genetics.118.301705
3.Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: Precision and plasticity via allostery. Nature Reviews. Molecular Cell Biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152
| None |