Part:BBa_K2912011

M. micrantha_leaves_ Unigene0029128

This is a single-stranded DNA that can be cyclized by T7 promoter, since it has two sites complementary to T7 promoter. It can transcribe RNA interference (RNAi) molecule by rolling circle transcription, which can silence the gene encoding chlorophyll A-B binding family protein AB80 of M. micrantha, through which we can block this essential matabolic gene expression to kill such invasive weed.

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]

SZU-China 2019 iGEM team

SZU-China 2019 iGEM team decides to synthesize the Micrancide, an RNAi-based herbicide for M. micrantha, to remove the weed by silencing the essential metabolic gene of it through RNA interference (RNAi) technology.

Usage and Biology

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. It involves multiple RNA-protein interactions characterized by four major steps:

1. Assembly of siRNA with the RNA-induced silencing complex (RISC)

2. Activation of the RISC

3. Target recognition

4. Target cleavage of mRNA

Hence, inspired by successful examples of RNAi technology, we decided to apply RNAi technology to the development of the herbicide for M. micrantha. It is a single-stranded DNA (ssDNA) that can be cyclized by the T7 promoter since it has two sites complementary to the T7 promoter (Fig.1). It can transcribe RNA interference (RNAi) molecule, ex. '''RNAi nanoparticles''', by rolling circle transcription.

Fig.1 Schematic representation of single-stranded DNA (ssDNA)

The RNAi nanoparticles are totally made of hairpin RNA, which are cleavable into countless siRNAs. This DNA sequence containing two regions that could be transcribed into a hairpin siRNA via T7 RNA polymerase, which would be cut into different kinds of siRNAs we need by Dicer enzyme inside the weed. We first phosphorylate the ssDNA and then cyclize the ssDNA to form a closed circle. Through the rolling circle transcription of the circle sequence, there were half-million of hairpin siRNA precursors linking together and packing to form a nanoball, the RNAi nanoparticle (Fig.2).

Fig.2 Schematic representation of self-assembled RNAi nanoparticles

The gel electrophoresis image of cyclized ssDNA and the RNAi nanoparticles are as follows (Fig.3, 4). Lane 5 of Fig.4 is this sequence’s GE image of RNAi nanoparticles. We can see that the cyclized ssDNA were trapped above the uncyclized one by 3% agarose gel. And the nanoparticles of large size were trapped at the beginning and the smaller ones ran down.

Fig.3 The gel electrophoresis image of cyclized ssDNA

Fig.4 The gel electrophoresis image of RNAi nanoparticles

Then we observed the morphology of the synthesized RNAi nanoparticles treated coated by gold (Au) under scanning electron microscope. We saw the nanoball with thousands of pleated sheet structures, which were the folded hairpin siRNAs (Fig.5).

Fig.5 The SEM image of RNAi nanoparticles

After synthesizing the RNAi nanoparticles of this DNA sequence, we tested the silencing efficiency of this RNAi nanoparticles, and this kind of RNAi nanoparticles can silence the gene encoding chlorophyll A-B binding family protein AB80 of M. micrantha. The qrt-PCR and apparent morphology changes results are as follows (Fig.6, 7). We can see that the relative gene expression of target gene dropped significantly after treated for one days, while the negative control almost had no change, which meant that the RNAi molecules worked to silence the gene expression as expected. Moreover, from the morphology of tested leaf, we can see that the leaf of M. micrantha turned brown and wilting after treated for 7 days.

Fig.6 The relative gene expression of gene 29128

Fig.7 The Morphology of the Leaves after Testing for 7 days

Moreover, we have tested the content changes of siRNAs cut from this RNAi nanoparticles introduced into the leaves through a G-quadruplex DNA-based, label-free, and ultrasensitive strategy (Fig. 8). We can see that the siRNA in vivo increased dramatically after treated for 1 days and then decreased gradually. For more information, please visit BBa_K2912016 or SZU-China 2019 iGEM Measurement.

Fig.8 The Fluorescence Emission change of differently treated siRNA samples

References

[1] Kupferschmidt, K. A Lethal Dose of RNA[J]. Science, 2013, 341(6147):732-733.

[2] Lee J B, Hong J, Bonner D K, et al. Self-assembled RNA interference microsponges for efficient siRNA delivery[J]. NATURE MATERIALS, 2012, 11(4):316-322.

[3] Yan L, Yan Y, Pei L, et al. A G-quadruplex DNA-based, Label-Free and Ultrasensitive Strategy for microRNA Detection[J]. Sci Rep, 2014, 4:7400.