Part:BBa_K3924031

miniSOG, mutagenesis from Arabidopsis phototropin 2

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 43
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 43
  Illegal NheI site found at 4
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 43
  Illegal BamHI site found at 37
  Illegal XhoI site found at 364
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 43
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 43
  
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: miniSOG
Base Pairs: 372bp
Origin: Arabidopsis thaliana
Properties: A mutagenesis phototropin protein which can generate ROS after being exposed to blue light

Usage and Biology

The miniSOG was engineered by-site specific mutagenesis from Arabidopsis phototropin 2, which naturally binds FMN but does not generate ROS ^[1]-[3]. By mutation, it can generate ROS after being exposed to blue light and ROS can oxidize guanosine into an intermediate, which could be attacked by primary amine to form covalent bond with NH2-containing probes which could be used to label RNA. In this way, we are able to label RNA molecule with biotin or fluorophore for further detection. Considering the accessibility of the probes, we used biotin-NH2 probes for our labelling assay.

Figure 1: The labelling principle of RNA using miniSOG^[1]

Design and Construction

For this part, we got the plasmid and the amino acid sequence thanks to a gift from the Peng Zou lab, Peking University. Then we directly used the plasmid for subsequent miniSOG tests.

Functional Verification

Validation based on protein expression level

After getting the synthetic plasmid, we test it by DNA sequencing and the result showed correctness of the sequence. Then we transformed the plasmid into BL21 chemocompetent cells and expressed miniSOG successfully. We first test the expression of miniSOG by Coomassie bright blue staining and the result is below (Fig. 2).

Figure 2: The Coomassie dyeing result

Validation based on protein activity

Then the purified miniSOG was used to test it labelling ability and the results of labelling strongly suggested the protein activity of miniSOG (Fig. 3). Under blue light illumination, miniSOG can catalyze labelling of RNA with biotin-NH2 probes. After the reaction, RNA Clean & Concentrato^TM-25 (ZYMO, Catalog #R1017, R1018) was used to get rid of excess biotin-NH2 probes and dot-plot assay was used for color development using streptavidin-HRP followed by substrates (see protocol for experimental details). This result is in line with the results reported by previous literature^[1], and we established a relatively fixed work flow for labelling assay during these initial experiments.

Figure 3: The non-specific labelling of RNA using miniSOG

To prepare for the subsequent test of specific markers, we tested the sensitivity of miniSOG RNA labelling ability after verifying its activity (Fig. 4). From this test, the result shows, when the RNA concentration is above 200 ng/ul, miniSOG can significantly label RNA. Therefore, the RNA concentration of 200 ng/ul is used as the standard line for detection in our later experiments.

Figure 4: The result of miniSOG RNA labelling sensitivity test

Reference

[1] Wang, P., Tang, W., Li, Z., Zou, Z., Zhou, Y., Li, R., Xiong, T., Wang, J., & Zou, P. (2019). Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling. Nat. Chem. Biol., 15(11), 1110–1119. https://doi.org/10.1038/s41589-019-0368-5
[2] Ding, T., Zhu, L., Fang, Y., Liu, Y., Tang, W., & Zou, P. (2020). Chromophore-Assisted Proximity Labeling of DNA Reveals Chromosomal Organization in Living Cells. Angew. Chem. Int. Ed., 59(51), 22933–22937. https://doi.org/10.1002/anie.202005486
[3] Ruiz-González, R., Cortajarena, A. L., Mejias, S. H., Agut, M., Nonell, S., & Flors, C. (2013). Singlet oxygen generation by the genetically encoded tag miniSOG. J. Am. Chem. Soc., 135(26), 9564–9567. https://doi.org/10.1021/ja4020524