Engineering Success

We engineered a fragment containing the p19gene of the tomato stunt virus with an added His-tag and a functional T7 promoter-driven expression cassette for a long dsRNA. The whole fragment was cloned into a pUC19 backbone. The engineering success of this construct was then validated by conduction of a western blot for p19 expression, NGS for sequence congruency and Native Page for siRNA presence. The functionality of the dsRNA expression cassette was validated by in vitro transcription using a commercial kit. To further enhance and regulate the production of p19, we added a Shine-Dalgarno and lacO sequence as addition to the tac-promoter-driven RNA transcription by primer extension PCR.

Methods

For detailed methods please refer methods for siRNA production.

Engineering of pUC19-p19-siRNA UL19

We initially simulated the folding of the chosen loop element in the dsRNA expression cassette with UNAFold (N. R. Markham & M. Zuker, 2008) by selecting a sequence spanning from the +1 site of the T7 promoter to the 20th base of the T7 terminator. Two possible folding structures with ΔGs of -31.40 kcal/mol (Structure 1, Figure 1A) and -30.60 kcal/mol (Structure 2, Figure 1B) were identified.

pUC19-p19-siRNA empty was engineered by restriction based cloning of a PCR amplified backbone and an insert obtained by solid phase synthesis. Primers for pUC19 backbone amplification were analysed on a temperature gradient ranging from 64 °C to 71 °C. The annealing temperature proposed by NEB T_m- Calculator (https://tmcalculator.neb.com/#!/main) was 72 °C. For all temperatures we obtained amplicons with a size ranging from 1500 to 2000 bp as well as a second fragment between 600 and 700 bp (Fig 2A). An annealing temperature of 69 °C was chosen and the PCR was repeated (Fig 2B). Plasmids received by plasmid preparation were sequenced with p19-forward and AmpR reverse. Insert presence was proven as well as integrity of the loop structure.

Here sequence alignments can be found for pUC19-p19-empty p19F and pUC19-p19-empty AmpR.

siRNA target areas were amplified with SacI/XhoI or SalI/NotI sequence extension by PCR. We obtained amplicons with a size ranging from 200 to 300 bp (Figure 2C) which was congruent with the expected sizes of 266 bp for the SacI/XhoI Primer set and 268 bp for the NotI/SalI primer set. After performing a two-step cloning and transformation in E. coli, we analysed the obtained plasmids by restriction digest with SacI and NotI to confirm the presence of the insert in both restriction cassettes. Results were visualised on a 1.2 % Agarose Gel stained with Ethidium Bromide (Fig 2D). We compared the resulting fragment sizes to the theoretical sizes obtained by digest in NEB Cutter v3 (Tab. 1).

Plasmids were sequenced with p19F and AmpR to confirm correct insertion and conservation of the loop structure as well as the integrity of p19 gene.

Here sequence alignments can be found for pUC19-p19-siRNA UL19 p19F and pUC19-p19-siRNA UL19 AmpR.

Subsequently we checked whether for the obtained sequence of the dsRNA expression cassette the formation of a loop structure is predicted. We chose a sequence from the +1 site of the T7 promoter to the 20th base of the T7 terminator to include the full expression cassette. Folding simulation resulted in one proposed structure with a ΔG of -605.90 kcal/mol (UL 19 dsRNA.pdf ).

We furthermore introduced a 7 bp long fragment between the tac promoter and the Startcodon of p19 by Primer Extension PCR.

Sequence Alignments for pUC19-p19-siRNA tac extension can be found: here.

After the successful cloning of pUC19-p19-siRNA UL19 in, we extend the insertion by a lac operator sequence and a Shine-Dalgarno sequence using the same principle. The lastly obtained plasmids were analysed by restriction digest with ClaI and HindIII on a 1.2 % Agarose Gel stained with Ethidium Bromide (Fig 2E). We compared the obtained fragment sizes to the theoretical sizes acquired by digest in NEB Cutter v3 (Tab. 2).

Afterwards the Plasmid was sequenced with p19F and AmpR to confirm correct insertion of both inserts and conservation of loop structure as well as integrity of p19 gene. Furthermore the plasmid was sequenced with pUC19-pBR322ori-fwd to confirm complete presence of tac-lacO-Shine Dalgarno structural element. The proposed plasmid structure is displayed in Figure 2F.

Sequence Alignments for pUC19-p19F-UL19 siRNA Tac-LacO-SD pBR322 fwd can be found: here.

Evaluation of pUC19-pp19-siRNA UL19 functionality

We assessed the functionality of our plasmid in two different ways. At first we checked for expression of p19 by SDS-PAGE and Western-Blot. To determine the protein concentration a Bradford Assay was conducted (for results see Table 6, Fig 4A). 30 µg of Protein in the Wash Fraction 1C and a linear mass dilution from 3 µg to 3 ng of Ni-NTA bead bound p19-fraction 1 were analysed by SDS-PAGE. Ponceau staining of the Blot revealed in each fraction an accumulation of a protein with a mass around 28 kDa. This protein is most dominantly present in Wash fraction C together with other proteins of different sizes. In the linear mass gradient only a small band of a ~28kDa protein is visible with decreasing intensity at lower concentrations. In comparison to the loaded BSA masses (1 µg and 500 ng) only in the Wash fraction 1C contains comparable amounts of protein. The linear gradient shows high purity of the fraction with only one visible protein (Fig 4C).

Functionality of the T7 expression cassette was assessed by in-vitro transcription. Our self- designed and cloned plasmid was compared to a cassette derived by solid phase synthesis and a PCR full amplicon of this solid phase synthesis derived cassette. Both cassettes derived from the solid-phase synthesis did not yield any visible dsRNA, while. Our cassette produced a dsRNA at a size of ~300 bp (see Figure 4B).

Production of pro-siRNA in E. coli T7 Express lysY/I^q

Production of pro-siRNA was analysed by native PAGE. This resulted in a methylene blue stained gel (Fig 4). For each fraction of 500 ng two different fragment patterns can be observed.: oOne above the positive control (siRNA 21 nts) and one slightly below the positive control. Fragments running roughly at the same height as the positive control are therefore between 18 and 21 nts in size. These were cut from the gel for further processing.

Discussion

We optimized the sequence provided to us by Huang and Lieberman by addition of a T7-Terminator and a rrnB-T1 Terminator to more tightly regulate the gene expression. This allowed us to produce a single dsRNA with a size of 300 bp. In contrast to a size range which would have been obtained when no T7-Terminator is present. In casae of p19 expression we noticed that our initially design lacked a functional p19 expression casette. We therefore, altered our design and inserted the missing sequence for tac-mediated transcription as well as a lac operator for transcriptional control to redulate the gene expression more tightly since the tac promoter is a constitutive promoter with high expression levels. To enhance ribosom binding we included a Shine-Dalgarno sequnce downstream of the lac operator to ensure that the Shine-Dalgarno sequence is present in the transcribed mRNA. Whilst the T7 dsRNA expression casette is highly functional the p19 expression and purfification process can be enhanced. Purification can be eased and more yield can be achieved by usage of a different tag which binds more selectivly to it's counterpart therefore reducing unselective binding which occurs usually Ni-NTA beads (compare Westernblot wash fraction 1C). Tags like Haemaglotinin or GST may yield purer fractions. Overall, the proposed expression system for pro-siRNAs in E. coli is functional and the obtained pro-siRNAs are capable to faciliate knockdown in-vitro (see proof of concept, results cellculture assays).