Team:OUC-R/Model/Futh




Theoretical Models

  • Z-HJ Element model

    This project intends to use a DNA double-loop structure with hair pins, with a total of 86nt and a Linking number of 6.

    The array is interconnected by connecting ZBP1, antibody and other inducers with the purine-pyrimidine sequence, where the array is connected by a pipe. That is, the LK of APP sequence changes from +1 to -1, and to ensure the Linking number of the system is 6, the promoter sequence on the hairpin structure changes to right helix, that is, the LK value of the hairpin structure changes from 0 to +2, and the hammerhead Ribozyme gene can be expressed normally.With the formation of the transcription bubble, APP sequence will change back to +1. The tightness of the inducer binding will affect the way of transcription. If the inducer binding is loose, rolling loop transcription may occur. By detecting the presence and concentration of hammerhead Ribozyme, we can predict the concentration of large detection substance and its binding ability with APP sequence. At the same time, we can increase the concentration of magnesium ion in the reaction condition, so that the z-DNA exists before the inducer is added, and then we can further study it. For comparison, we also replaced the APP sequence with a random sequence and conducted the same experiment. And we tested the formation of the double ring structure at different temperatures to find its optimal conditions.

  • Regulation of Z-HJ elements in intracellular environment

    Because the environment inside the cell is more complex than the environment outside the cell. Therefore, we need a stable intracellular environment to verify the regulatory role of our Z-HJ element.

    • E3L protein

      Poxvirus gradually develops a variety of strategies against interferon reaction during its interaction with the host, so as to destroy or evade the antiviral effect of interferon. E3 protein is an early non-structural protein encoded by vaccinia virus (VV)E3L gene, which consists of z-DNA binding domain (ZDBD) located at the amino terminal and dsRNA binding domain (DRBD) located at the carboxyl terminal. E3L protein is the smallest protein in the Z-DNA binding protein family, so we used it as an inducer for the Z-HJ element.

    • 3-WJdB

      In vitro we detect the expressed products by expressing RNA enzymes, but in vivo we want to detect the expressed products of our components with a visual and simple detection method. After consulting OUC-R, another team in our school, we found that their 3WJdB is a simple, effective and non-toxic method for detection. So we turned to their team to build our test. The expression of 3WJdB is regulated by the Z-HJ element on the promoter. When 3WJdB is expressed, it can bind to DFHBI and emit fluorescence for the detection of cells.

    • Recombinant E. coli

      Z-hj elements were detected by recombinant EScherichia coli. The E3L homologous gene of sheep poxvirus was cloned using sheep poxvirus as template. The gene is 534 bp and can encode 177 amino acids. The recombinant plasmid PGEX4T-1-SPPV E3L was constructed and transformed into DE3 receptor cells to construct recombinant EScherichia coli.

    • E3L protein induces expression of Z-HJ element

      Our Z-HJ element was introduced into the recombinant E. coli, and E3L protein was used as z-DNA binding protein to induce APP sequence B-DNA to be transformed into Z-DNA. Through negative superhelical stress, cross structure was opened and promoter sequence was exposed to express 3WJdB, and then detection was conducted.

  • The Z-HJ element was used to detect the expression level of ADAR-1

    Adar-1 protein :ADAR-1 protein is a double-stranded RNA-specific adenosine deaminase, which is widely present in a variety of organisms from nematodes to humans. Adar-1 is the largest protein in the Z-DNA binding protein family, with two Zα binding domains at the N-terminus. Our Z-HJ element can be used as an ADAR-1 sensing element to discover the regulatory effect of ADAR-1 on unconventional DNA transcription, and even to detect the expression level of ADAR-1.




Furthermore

In order to explore whether there is similar app-cross sequence structure in organisms, we used software (part of the software is connected here) to explore the structure of app-cross sequence. We respectively selected THE DNA sequences of E. coli and human for search, and the specific steps are as follows:

  • - Search for stem-loop sequences that match our expectations

  • - Record the position of stem-loop sequences

  • - Search for a corresponding APP sequence ahead of the position

  • - Record and make statistical tables

In the selection of data, we specifically searched for:

  • - stem-loop sequence：the length of stem between 5bp-20bp, and the length of loop between 2bp-4bp.

  • - App sequence: more than 10bp.

And finally, we have：

  • As for E.coli：

  • As for human：

These data support the idea of a new regulatory mechanism within cells. Next, we will search for promoters and genes near the Z-HJ elements in these bodies, and further verify the possibility that these elements regulate gene expression.




References

[1]Yeon-Mi Lee, Hee-Eun Kim, Eun-Hae Lee, Yeo-Jin Seo, Ae-Ree Lee, Joon-Hwa Lee. NMR investigation on the DNA binding and B–Z transition pathway of the Zα domain of human ADAR1. Biophysical Chemistry 2013, 172 , 18-25. 

[2]D.-B. Oh, Y.-G. Kim, A. Rich.Z-DNA-binding proteins can act as potent effectors of gene expression in vivo.Proc Natl Acad Sci U S A, 99 (2002), pp. 16666-16671

[3]Dina Zhabinskaya* and Craig J. Benham Competitive superhelical transitions involving cruciform extrusionNucleic Acids Res. 2013 Nov; 41(21): 9610–9621.