Part:BBa_K5055002

Biology

P21 is one of the 9 proteins translated by the BYV. It weights 21 kDa and has a role of suppressor for silencing RNA. It is considered as a nucleic acid protein that binds to small interfering RNA (siRNA). Its mechanism is quite simple: it binds and isolates the siRNA from its loading into the RNA Induces Silencing Complex (RISC). Moreover, it has been shown to be intrinsically linked with BYV viral amplification.

Design of the part

After some bibliographic research, we looked for the coding sequence of the p21 protein of the Beet Yellows virus (BYV). This protein acts as a suppressor of double-stranded RNA silencers and is part of a new family of RNA silencing suppressor present in Closterovirus (the family of the BYV). Eliminating this protein will allow the upholding of our siRNAs, and as well the destruction of a part of the virus genome. We kept a sequence of 408 bases from the sequence of the p21 of the BYV, found on NCBI (GenBank: U71297.1).

We had trouble synthesizing these sequences because DNA manufacturers are not able to synthesize sequences with repeats over 19 bp. Therefore, we decided to divide our sequences into two: one encoding the sense sequence and the other encoding the antisense sequence of our hairpin RNA. To be able to assemble these two parts, we chose our loop sequence so that it contained the restriction site of the enzyme NdeI.

The loop structure allows a better detection of the lhRNA by the Dicer complex. It also confers better stability to the solution, compared to double-stranded RNA. We needed to incorporate a few bases providing a loop configuration between sense and antisense sequences of the chosen targets. To design the loop, we saw in the literature that for lhRNA designs, we should use loop sequences of around 8 nucleotides for a final size of around 1kb, with a C and a G base at the extremities of the loop for increased stability. It then requires a random mismatch for the loop opening.

We tried the in silico-functioning of our construction thanks to the RNAfold WebServer: http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi .

In addition, we wanted to protect our lhRNAs by encapsulating them in the capsid of the Tobacco Mosaic Virus (TMV). Therefore, we added the Origin of Assembly Sequence (OAS) of the TMV at the end of the lhRNA sequence. This sequence is recognized by the coat proteins of the TMV, and this allows the formation of the capsid around a nucleotide sequence.

The plasmid encoding lhRNAs will be introduced into DH5α E. coli bacteria, known for plasmid amplification. They have important transformation yields, deficiency in recA1 avoiding clone rearrangements or degradation by recombination and ensuring their stability.

Usage

We plan to transform the complete plasmid containing this lhRNA sequence in E. coli strain HT115, in order to produce the lhRNAs. This strain is deficient for RNase III which usually recognizes and cleaves double-stranded RNA. Moreover, the HT115 strain also produces the T7 RNA polymerase, when cultivated with IPTG, that has a very high rate of transcription. This will allow a higher production of the lhRNAs.

Encapsulation

We aimed to assemble the lhRNAs in the coat proteins. To do so, we planned to mix the lhRNAs and the coat proteins. Thanks to the OAS sequence mentioned above, the coat proteins will recognize it and will wrap around the lhRNAs naturally, allowing their encapsulation.

Implementation

When the lhRNAs are encapsulated, we plan to spray them directly on sugar beet leaves, before or after BYV infection. The capsid will automatically be degraded and allow the entry of the lhRNAs in the cells. They will then be processed by the RNA interference machinery of the plant cell. Finally, the resulting siRNAs will recognize the BYV genome and allow its degradation thanks to the RISC.

Characterization

Since we divided our sequence into two parts, we needed to ligate these two together to form the basic part. The result of this ligation is presented on our wiki page.

In the gel, we saw that we obtained the expected bands. In fact, there are bands at around 1000bp corresponding to the two inserts ligated together and bands at around 500bp corresponding to the insert alone and not ligated. It is coherent with the expected results since the p21 lhRNA insert is 1020bp long. The sense insert is 454 bp long and the antisense 584.

We did not manage to ligate the constructed basic part with the backbone of interest. We will therefore have to perform the ligation before conducting the next steps.