We adapted a method for production of recombinant siRNAs in E. coli published by Huang et al. in 2013 (Huang et al., 2013; Huang & Lieberman, 2013).
To evaluate our chosen method in a simple and efficient way, the EGFP gene was chosen as a proof-of-concept target for our liposomal siRNA delivery system. We used the pEGFP-C1 plasmid (Addgene No. 2487) containing the EGFP gene published by Yang et al. (Yang et al., 1998). We then selected the gene UL19 encoding for HSV-1 major capsid protein for evaluation of potential HSV treatment methods using liposomal formulations.
The sequence of UL19 encoding for HSV-1 major capsid protein was extracted from the complete human herpesvirus 1 genome. The sequence was human codon optimised using Benchling.
The sequence was screened for published antibody binding sites and functional structure elements using the UniProt database. We identified the amino acids 862 to 880 in UL19 as a published antibody binding site (Han et al., 2019). Since it is a published antibody site, we anticipate that the corresponding DNA sequence is highly conserved in the HSV genome. Therefore, we chose a sequence for cloning and expression including this structure element. For UL19 bases 2446 to 3444 were selected resulting in a fragment of 999 bp in size. The fragment was further modified to mask unwanted restriction sites and ease primer design while keeping the amino acid sequence unchanged. UL19 was modified at the 16-18TCC>AGT and 21G>A. A His-tag (5’-CATCACCATCACCATCAC-3’) and a TAA stop codon were added at the 3’ sequence end. The modified sequence was synthesised via Eurofins Genomics gene synthesis service and delivered cloned in a pEX-A258 vector.
Huang et al. recommend that the siRNA target area’s size should range between 250 and 500 bp (Huang et al., 2013). We selected a 249 bp area for UL19 (Sequence: 2527 – 2775; Fragment: 82 - 330). The siRNA target area includes the previously described structural motif. For EGFP knockdown a previously published siRNA sequence (sense: 5’-GCAAGCUGACCCUGAAGUUCAUTT-3’; (Metwally, A. A., Blagbrough, I. S., & Mantell, J. M., 2012) was chosen.
The analysis of our target areas with siRNA Design Tool from IDT revealed 13 potential siRNA candidates for UL19. 2 of these 13 candidates were indicated as cross-reacting with human gene transcripts.
100 ng of pEX-A258-HSV-UL19-6xHis and pEGFP-C1 were transformed into competent E. coli DH5α (New England BioLabs) according to manufacturer’s protocol. pEX-A258-HSV-UL19-6xHis transformants were plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight. pEGFP-C1 transformants were plated onto LB-Kan plates (75 µg/mL Kanamycin) and incubated at 37 °C overnight. Subsequently they were inoculated into 5 mL LB-Amp (100 µg/mL Ampicillin) or LB-Kan (75 µg/mL Kanamycin) media and incubated at 37 °C at 180 rpm overnight. The plasmids were isolated from 4 mL of overnight culture with the QIAPREP Spin Miniprep Kit. For quality control, 500 ng of each plasmid were digested with 10 units of EcoRI-HF (New England BioLabs) in a total volume of 30 µL and analysed on a 1.2 % TAE-Agarose gel stained with ethidium bromide.
100 ng of pRK5 were transformed into competent E. coli NEB10-beta (NEB). The pRK5 transformants were plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight before subsequently being inoculated into 5 mL LB-Amp (100 µg/mL Ampicillin) and incubated at 37 °C at 180 rpm overnight. Plasmids were then isolated from 4 mL of the overnight culture with the QIAPREP Spin Miniprep Kit. For quality control 500 ng of each plasmid were digested with 10 units of EcoRI-HF (New England BioLabs) in a total volume of 30 µL and analysed on a 1.2 % TAE-Agarose gel stained with ethidium bromide.
The congruence of the sequence was confirmed by NGS for pRK5 and pEGFP-C1 using CMV-Forward and SV40A-Reverse.
A missing Kozak-sequence was inserted to enhance the translation of HSV UL19 AA816:1148-6xHis via PCR. The HSV UL19 fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV UL19-rev. and roughly 1 ng of pEX-A258-HSV-UL19-6xHis, the total reaction volume being 20 µL, for this purpose. The success of the PCR was evaluated on a 1.2 % TAE-Agarose gel stained with ethidium bromide. Successful PCR products were pooled and purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using Nanodrop 2000.
| Primer | Sequence | Tm [°C] | TPCR [°C] |
| pRK5-Kozak-fwd. | 5’-CCAACTGCACCTCGGTTCTATCGATTGAATTCGCCACCATGGTGCCTGCGTTCAGTCGAG-3’ | 72.1 | 68 |
| HindIII-HSV UL19-rev. | 5’-ATCCAAGCTTTTAGTGATGGTGATGGTGATGTCGT-3’ | 63.5 | 68 |
500 ng of the obtained PCR product and pRK5 were each digested with 10 units of EcoRI-HF and 10 units of HindIII-HF (NEB) for 1 h at 37 °C separately. The restriction digests were then purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using Nanodrop 2000.
The fragments were ligated at a 3:1 (insert:vector) molar ratio. The 65.22 ng of UL19 insert were thus ligated with 100 ng of
pRK5 vector using T4 ligase (Jena Biosciences) in a total reaction volume of 20 µL (supplied with 2 µL 10x Standard Ligation Buffer)
at 16 °C for 30 min. After that, the ligase was inactivated at 65 °C for 15 min and chilled on ice afterwards for 5 min. 5 µL of the
ligation mix were transformed into NEB 5α competent E. coli (NEB) according to manufacturer’s protocol, plated onto LB-Amp plates
(100 µg/mL Ampicillin) and incubated at 37 °C overnight. 12 clones were picked,inoculated into 5 mL LB-Amp (100 µg/mL Ampicillin) and
incubated at 37 °C at 180 rpm overnight. The plasmids were isolated from 4 mL of the overnight culture with the QIAPREP Spin Miniprep Kit
according to the manufacturer's protocol. For quality control 500 ng of each plasmid were digested with 10 units of EcoRI-HF
(New England BioLabs) and 10 units of HindIII-HF (NEB) in a total volume of 30 µL and analysed on a 1 % TAE-Agarose gel stained with
ethidium bromide.
The congruence of the sequence was confirmed by NGS for pRK5-HSV UL19 AA816:1148-6xHis using CMV-Forward and SV40A-reverse primer.
We designed a construct containing two expression cassettes. One for p19-6x His expression and one for expression of prä-pro-siRNA. The p19 sequence was a gift from Linfeng Huang and Judy Lieberman at the Boston Children’s Hospital and is congruent with the sequence of Addgene (cat. no. 46306).
The p19 gene is flanked by a tac-promotor and a rrnBT1 transcription terminator. The empty siRNA expression cassettes are flanked by a T7-promotor and a T7 transcription terminator. The first siRNA cassette is located between the SacI and XhoI restriction site. The second is between the SalI and NotI restriction sites. Both cassettes are connected by a 32 bp long linker sequence facilitating the folding of ss-pre-pro-siRNA to ds-pre-pro-siRNA (5’- TCTAGAGCGCACGTAtacACGCGCTGATCAGC-3’) after transcription. The whole construct is flanked by non-coding backbone sequences of pUC19 containing ClaI restriction site at 5’-end and BamHI restriction site at 3’-end. The sequence was ordered at Twist Bioscience. During the evaluation of the full plasmid we noticed missing p19 expression and therefore, redesigned the tac-promoter site using primer extension PCR. We increased the spacer length as published by Mulligan, M. E., Brosius, J., & McClure, W. R. (Mulligan, M. E., Brosius, J., & McClure, W. R., 1985), inserted a lacO element and added a Shine-Dalgarno sequence. The registry part BBa_K2172009 served as the design template.
pUC19-p19 6xHis-siRNA empty was produced by restriction based cloning of PCR amplified pUC19 Backbone and p19-siRNA empty construct. PCR was performed using Phusion 2x Master Mix with HF Buffer (New England BioLabs) with 0.5 µM forward primer and 0.5 µM reverse primer and around 1 ng template in a total reaction volume of 20 µL. Success of PCR was validated on 1.2 % agarose gel stained with ethidium bromide.
| Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| pUC19-BamHI-fwd. | 5’-TCCGGATCCGAAACGCGCGAGACGAAAGGG-3’ | 68.9 | 72.0 | 69.0 |
| pUC19-ClaI-rev. | 5’-ATCCATCGATTCAGGGGATAACGCAGGAAAGAACA-3’ | 64.4 | 72.0 | 69.0 |
| p19-BamHI-rev. | 5’-GGATGGATCCGCAAAAGGCCAG-3’ | 60.9 | 69.0 | 68.0 |
| p19-ClaI-fwd. | 5’-ATTATCGATAACGCCAGCAACGCG-3 | 59.9 | 69.0 | 68.0 |
The PCR products were pooled, purified with QIAGEN PCR Clean Up Kit and concentration was measured with Nanodrop 2000. 500 ng of each PCR was digested with 10 units BamHI and 10 units ClaI (NEB) for 1 h at 37 °C in 50 µL. Samples were recovered with QIAGEN PCR Clean Up Kit and concentration was measured with a Nanodrop 2000.
The fragments were ligated at a 3:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (Jena Bioscience) at 23 °C for 30 min in a total volume of 20 µL in duplicates. 5 µL of each ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.
Of each transformation four positive clones were picked and inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. The plasmids were isolated from 4 mL of each overnight culture with QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted into 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.
For quality control, restriction digestion was performed. 500 ng plasmid was digested with EcoRI-HF (NEB) to assure correct size and insert presence. It was analysed on 1.2 % TAE-Agarose gel stained with ethidium bromide.
The construct was sequenced with p19-Seq.-fwd. and pUC19-AmpR-rev.
Designated siRNA target area was amplified by PCR using two different primer sets. pEX-A258-UL19-6xHis served as template. PCR was performed using Phusion 2x Master Mix with HF Buffer (New England BioLabs) with 0.5 µM forward primer and 0.5 µM reverse primer and ~1 ng template in a total reaction volume of 20 µL. The success of the PCR was validated on 1.2 % agarose gel stained with ethidium bromide. PCR products were pooled, purified with QIAGEN PCR Clean Up Kit and their concentration was measured with Nanodrop 2000.
| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| BBa_K4344055 | SacI-HSV-UL19-6xHis-fwd. | 5’-ATGAGCTCGTCGTACCTGAGATTGCTCCAG-3’ | 64.0 | 72.0 | 65.0 |
| BBa_K4344075 | XhoI-HSV-UL19-6xHis-rev. | 5’-ATCTCGAGTGTTAGTGGTAGAGGCGGTGTTG-3’ | 63.7 | 72.0 | 65.0 |
| BBa_K4344033 | NotI-HSV-UL19-6xHis-fwd. | 5’-ATGCGGCCGCGTCGTACCTGAGATTGCTCCAG-3’ | 70.0 | 72.0 | 65.0 |
| BBa_K4344059 | SalI-HSV-UL19-6xHis-rev. | 5’-ATGTCGACTGTTAGTGGTAGAGGCGGTGTTG-3’ | 64.0 | 72.0 | 65.0 |
500 ng of pUC19-p19 6xHis-siRNA empty and 1 µg UL19 siRNA target PCR Product obtained by primer set 1 were digested with 20 units of SacI-HF and XhoI in CutSmart buffer in a total volume of 50 µL at 37 °C for 2 h. Products were recovered with the QIAGEN PCR Clean Up Kit and concentrations were measured with Nanodrop 2000.
Fragments were ligated at a 5:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (Jena Bioscience) at 16 °C for 30 min in a total volume of 20 µL. 5 µL of the ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight. Of each transformation four positive clones were picked, inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. Plasmids were isolated from 4 mL of each overnight culture with the QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted into 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.
500 ng of a positive clone and 1 µg of UL19 siRNA target PCR product obtained by primer set 2 were digested with 20 units of NotI-HF and SalI-HF in CutSmart buffer in a total volume of 50 µL at 37 °C for 2 h. Products were recovered with the QIAGEN PCR Clean Up Kit and concentrations were measured with Nanodrop 2000.
Fragments were ligated at a 5:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (NEB) at 4 °C overnight in a total volume of 20 µL. 5 µL of each ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.
Of each transformation six positive clones were picked, inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. Plasmids were isolated from 4 mL of each overnight culture with the QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted using 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.
For quality control 500 ng of plasmid was digested with 20 units SacI and 20 Units XhoI, 20 units NotI-HF and 20 units SalI-HpF, 20 units SacI-Hf and NotI-HF to assure correct size and presence of both inserts. Results were analysed on 1.2 % TAE-Agarose gel. Positive clones were sequenced for presence of loop structure with p19-fwd. and pUC19-AmpR-rev.
We inserted a 7 bp long fragment by primer extension PCR between the tac promoter and the p19 starting codon using the primers sets pUC19-ori-fwd./tac-tac Extension-rev. and p19-tac Extension-fwd/p19-HindIII-rev in two different PCR reactions as described earlier with pUC19-p19-UL19 siRNA serving as template. Success of PCR was validated on 1.2 % TAE-Agarose gel stained with ethidium bromide. All successful PCR products were pooled and recovered with the QIAGEN PCR Clean Up Kit and concentration was measured with Nanodrop 2000. Both products were used as a template at equimolar ratios for ligation of both fragments by PCR using the primers pUC19-ori-fwd. and p19-HindIII-rev. Results were validated on 1.2 % TAE-Agarose gel. The PCR product with the correct size (502 bp) was extracted from the gel and recovered with the Qiagen Gel Extraction Kit.
| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| BBa_K4344052 | pUC19-pBR322-ori-fwd | 5’-GGGAAACGCCTGGTATCTTT-3’ | 55,1 | 63.0 | 63.0 |
| BBa_K4344071 | Tac-Tac Extension-rev. | 5’-TTTCCTTGTATAGCTCGTTCCATCACCACACCATTATACGAGCCGATGATTAATTGTCAA-3’ | 67.5 | 63.0 | 63.0 |
| BBa_K4344039 | p19-Tac Extension-fwd. | 5’-TTGACAATTAATCATCGGCTCGTATAATGGTGTGGTGATGGAACGAGCTATACAAGGAAA-3’ | 67.5 | 65.0 | 63.0 |
| BBa_K4344037 | p19-HindIII-rev | 5’-AGTGAAGCTTCCGTCCTGTC-3’ | 56.9 | 65.0 | 63.0 |
| BBa_K4344052 | pUC19-pBR322-ori-fwd. | 5’-GGGAAACGCCTGGTATCTTT-3’ | 55.1 | 63.0 | 63.0 |
| BBa_K4344037 | p19-HindIII-rev. | 5’-AGTGAAGCTTCCGTCCTGTC-3’ | 56.9 | 63.0 | 63.0 |
500 ng of the obtained PCR product and pUC19-p19-UL19 siRNA were each digested with 10 units ClaI and 10 units of EcoRI (NEB) for 1 h at 37 °C separately. The restriction digests were purified using the Qiagen Gel Extraction Kit. The concentration was measured using Nanodrop 2000.
The fragments were ligated at a 3:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (Jena Bioscience) at 16 °C for 30 min in a total volume of 20 µL. 5 µL of the ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight. Of each transformation four positive clones were picked, inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. Plasmids were isolated from 4 mL of each overnight culture with the QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted into 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.
Process was repeated for insertion of LacO and Shine-Dalgarno Sequence using primer sets pUC19-ori-fwd./tac-LacO-SD-rev. and tac-LacO-SD-p19-fwd./HindIII-p19-rev with the previously produced plasmid served as template. For ligation of both fragments pUC19-ori-fwd. and p19-HindIII-rev. were used.
The quality control was conducted by restriction digest of 500 ng plasmid with HindIII and ClaI and subsequent analysis on a 1.2 % Agarose gel. Sequence congruence was assessed by NGS with pUc19-ori-fwd., p19-fwd. and AmpR-rev.
100 ng of pUC19-p19 6xHis-UL19 siRNA was transformed into NEB high efficiency T7 Express lysY/Iq competent E. coli (NEB C3016I) according to manufacturer’s protocol. Transformants were plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.
One positive clone was selected, inoculated into 300 mL LB-Amp (100 µg/mL Ampicillin) and incubated at 37 °C and 180 rpm. OD600 was measured every hour with a conventional photometer. After OD600 reached 0.4 to 0.6 protein expression was induced by addition of 300 µL of 500 mM IPTG (0.5 mM final concentration). The cells were incubated at 37 °C for 2 h. Biomass was harvested by centrifuging at 3200 g, 4 °C for 45 min. The produced pellets were resuspended in 10 mL cold lysis buffer (50 mM phosphate buffer (pH 7.5), 300 mM NaCl, 10 mM Imidazole, 1 % (vol/vol) Triton X-100, 1 mg/mL lysozyme), transferred into a 50 mL Falcon tube and incubated at 4 °C on a rocking incubator for 1 h. Afterwards, the lysate was sonicated with an OmniRuptor Pulse for two minutes at 30 % power with ten second intervals on ice. The lysate was centrifuged at 4000 g, 4°C for 45 minutes. Supernatant was filtered clear using a 0.45 µm followed by a 0.22 µm syringe filter into a 15 mL falcon tube. 1 mL of Ni-NTA beads were added to the supernatant and the mixture was incubated at 4°C on a rocking incubator for 12 h. Ni-NTA beads were washed by centrifuging at 500g, 4°C for 10 minutes and removing the supernatant. 5 mL of fresh lysis buffer (without lysozyme) was added and beads were incubated at 4 °C on a rocking incubator for 10 minutes. This was repeated three times.
The purified p19 protein was separated from the beads by addition of 500 µL of lysis buffer (1 % SDS, 1 mM EDTA, 250 mM Imidazole in PBS pH 7.4) and centrifugation at 500g for 5 min at room temperature. The purified protein was then incubated on ice for 10 min to precipitate excess SDS present in the lysis buffer. The solution was centrifuged clear at 16000 rpm at a temperature of 4 °C for 15 min.
The protein amount in the purified p19 protein fraction was assessed by a modified Bradford-Assay published by Grinztalis et al.. A BSA standard curve ranging from 10 µg to 100 µg was established. The lysates were diluted to a total protein amount of 50 µg in 10 µL and prepared for the SDS-PAGE. 10 µL of a 2x Laemmli-buffer were added to each sample and heated at 95 °C for 10 min. The samples were then allowed to cool down at room temperature. All samples were loaded onto a 12 % Tris-SDS-PAGE. A loading control of 5 µL prestained protein ladder was loaded as a size standard. The gel was run at 110 V for 1 ½ h on ice. The proteins were extracted from the gel by semi dry Western Blotting on a nitrocellulose membrane at 15 V for 30 min. The blot membrane was blocked with 5 % milk powder in TBS-T for 1 h. Afterwards the blot was incubated with primary antibody Anti-His-tag mouse monoclonal antibody (1:10000, Proteintech) in a blocking solution, supplied with 10 mM sodium azide at 4 °C overnight. Secondary antibody solution (1:5000, Proteintech) in blocking solution supplied with 10 mM sodium azide at 4°C for two hours. The membrane was washed five times for 3 min to remove unselective bound antibodies. After that, the membrane was stained with DAB to detect the p19 protein. The images were analysed using a ChemiDoc XRS+ (BioRad Laboratories).
To assess the functionality of our T7-driven expression cassette HiScribe-T7 transcription kit from NEB was used. 100 ng of linearized pUC19-p19-UL19 siRNA plasmid were in a reaction according to manufacturer’s protocol in a total volume of 20 µL and incubated at 37 °C overnight. An aliquot was taken from the crude sample. The DNA template was removed by a DNase I digest at 37 °C for 10 min. This was followed by the clean-up with Monarch RNA clean-up kit (NEB). An aliquot was taken from the purified sample for comparison.
100 ng of pUC19-p19 6xHis-UL19 siRNA was transformed into NEB high efficiency T7 Express lysY/Iq competent E. coli (NEB C3016I) according to manufacturer’s protocol. The resulting transformants were plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.
One positive clone was selected and inoculated into 100 mL LB-Amp (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm. OD600 was measured every hour with a conventional photometer. After OD600 reached 0.4 to 0.6 production was induced by addition of 100 µL of 500 mM IPTG (0.5 mM final concentration). The cells were incubated at 20°C overnight.
Biomass was harvested by centrifuging at 3200 g, 4°C for 45 min. The resulting pellets were resuspended in 10 mL cold lysis buffer (50 mM phosphate buffer (pH 7.5), 300 mM NaCl, 10 mM Imidazole, 1 % (vol/vol) Triton X-100, 1 mg/mL lysozyme), transferred into a 50 mL falcon tube and incubated at 4 °C on a rocking incubator for 1 h. Lysate was sonicated with an OmniRuptor Pulse for two minutes, at 30 % Power with ten second intervals on ice. Lysate was centrifuged at 4000 g, 4°C for 45 minutes. Supernatant was filtered clear using a 0.45 µm followed by a 0.22 µm syringe filter into a 15 mL Falcon tube. 1 mL of Ni-NTA beads were added to the supernatant and the mixture was incubated at 4 °C on a rocking incubator for 12 h.
Ni-NTA beads were washed by centrifuging at 500g, 4°C for 10 minutes, and removing the supernatant. 5 mL of fresh lysis buffer (without lysozyme) was added and beads were incubated at 4 °C on a rocking incubator for 10 minutes. This was repeated three times.
Ni-NTA beads were resuspended in 500 µL of SDS buffer (50 mM phosphate (pH 7.5), 100 mM NaCl and 0.5 % (wt/vol) SDS) and incubated at 700 rpm on an Eppendorf Mixer for 10 minutes. Beads were pelleted by centrifugation at 500 g for 1 min and supernatant was transferred in a fresh 1.5 mL tube. This procedure was repeated once and the obtained supernatants were combined. Total amount per production 1 mL.
100 µL of cold 2 M KCl was added and the mixture was incubated on ice for 10 min. The precipitate was pelleted by centrifugation for 5 min at 16000 g, 4 °C. The supernatant was split into two samples containing around 500 µL and 50 µL of 3 M sodium acetate. 550 µL of isopropanol and 10 µg glycogen were added to precipitate pro-siRNAs. Samples were incubated at -20 °C for 2 h.
pro-siRNAs were pelleted through centrifugation at 16000 g, 4°C for 30 min. Supernatant was discarded and the pellet was washed with 1 mL of ice-cold 75 % (vol./vol.) ethanol. Supernatant was carefully removed. Pellets were air-dried for 5 min and resuspended in 100 µL of nuclease-free water. Concentration was measured with IMPLEN Nanophotometer.
15 % (wt/vol) TBE-PAGE was used for separation of pro-siRNA. 500 ng of pro-siRNAs were mixed with 1 volume of gel loading buffer. Gel was run at 150 V until bromophenol blue line was 1 cm from the bottom of the gel.
The gel was stained with methylene blue staining solution (0.2 % methylene blue in 0.4 M Sodium acetate : 0.4 M acetic acid (1:1)) for 15 min. Background staining was removed by incubation in distilled water. The pro-siRNA bands (~21-25 nts) were identified and cut from the gel.
Gel fragments were cut into pieces, transferred into a 1.5 mL tube and incubated in >500 µL 0.3 M NaCl at 4 °C on a rocking incubator overnight. 500 µL of isopropanol (500 µL isopropanol per 500 µL 0.3 M NaCl) and 10 µg glycogen were added to precipitate pro-siRNAs. The samples were incubated at -20 °C for 2 h. pro-siRNAs were pelleted through centrifugation at 16000 g, 4°C for 30 min. The supernatant was discarded and the pellet was washed with 1 mL of ice-cold 75 % (vol./vol.) ethanol. Any remaining supernatant was carefully removed. The pellets were air-dried for 5 min and resuspended in 100 µL of nuclease-free water. Concentration was measured with Nanodrop 2000.
The lipids and cholesterol were weighed in a 25 mL volumetric flask. The flask was filled up with CHCl3:MeOH (9:1) and shaken until the lipid was dissolved. 4 mL aliquots were stored at -20 °C until usage. For cholesterol the end concentration was set to 100 mM, for mPEG2000 10 mM, for DOTAP 46 mM and for DOPE 42 mM.
The lipid stocks were thawed to room temperature and volumes pipetted together in ratios set to the desired composition (mPEG:cholesterol:DOTAP:DOPE, 0.3:2:1:1), which can be seen in Table 5. For the 1 mM and 0.185 mM lipid films 56 µL and 91 µL methanol was added to the lipid solution. After mixing the lipid stocks, they were heated in a heating block to 50 °C and steamed with N2 gas for 15 to 45 minutes until the organic solvent had evaporated. After that step, the vial was dried under vacuum for 1 hour. The final lipid films were stored at -20 °C with the lid taped shut with parafilm.
| Final liposome concentration | 100 mM | 1 mM | 0.185 mM |
| mPEG2000 (10 mM stock) | 139.4 µL | 14 µL | 3 µL |
| Cholesterol (100 mM stock) | 93.0 µL | 9 µL | 2 µL |
| DOTAP (46 mM stock) | 101.1µL | 10 µL | 2 µL |
| DOPE (42 mM stock) | 110.8 µL | 11 µL | 2 µL |
Dual centrifugation is used in combination with SiLibeads to rehydrate and break the lipid film into liposomes. We compared two protocols: two step centrifugation and three step centrifugation. For each protocol the lipid film had to be thawed to room temperature.
Two step dual centrifugation (2-fold DC):
SiLibeads (type SL) were added to the lipid film together with the first volume of PBS and centrifuged for 15 min at 2500 rpm
in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The amount of PBS volume was calculated with the following
formula. If the formula gives a volume below 2 µL, 2 µL PBS should be used instead.
The vial was removed from the centrifuge and the second volume of PBS was added. It was centrifuged for 5 min at 2500 rpm in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The PBS volume was calculated with the following formula. If the formula gives a volume below 2 µL, 2 µL PBS should be used instead.
Three step dual centrifugation (3-fold DC):
This protocol was only used for DNA and RNA loaded liposomes. 2 µL of DNA or siRNA (containing 3 µg of oligonucleotides)
in PBS were added to the lipid film and the vial was centrifuged for 15 min at 2500 rpm in the Zentrimix 380 R at 20 °C with
acceleration of 5 and deceleration of 9.
The vial was removed from the centrifuge and SiLibeads (type SL) were added. It was centrifuged again for 15 min at 2500 rpm in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9.
The vial was removed from the centrifuge and 2 µL PBS were added. It was centrifuged for a third time for 5 min at 2500 rpm in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The vial was removed and 96 µL PBS were added. The vial was vortexed.
In the protocol published by Hirsch et al. in 2008, dry lipids were dissolved in ethanol. Since we had already solved our lipids in CHCl3:MeOH (9:1), we pursued two different ways we could implement their procedure.
For testing whether alternative solvents made a difference in liposome production, lipids solved in CHCl3:MeOH and lipids solved in ethanol were used. To extract the lipids for the ethanol experiment, the CHCl3:MeOH dissolved lipids were dried under N2 and solved in 400 µL ethanol. Lipid solutions with a total lipid mass of 24 mg were created with both lipids solved in ethanol and lipids solved in CHCl3:MeOH (59.5 µL mPEG, 288.7 µL cholesterol, 173.7 µL DOTAP and 178.6 µL DOPE). After pipetting the volumes in the respective tubes, the lipid solutions were stripped of the solvent under N2 and finally put under vacuum for 90 min at 35 mbar. The respective dry lipid mixtures can be stored in the fridge.
Dry lipid mixtures were thawed and 36 µL of PBS added to reach a final batch weight of 60 mg. Beads were added in a 1:1 ratio to the mass (60 mg beads). The samples were incubated at room temperature for 10 min and afterwards centrifuged for 30 min at 2500 rpm in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The vial was removed from the centrifuge and 120 µL PBS (200 % PBS in relation to VPG) were added. It was centrifuged for 30 s at 2500 rpm in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. Samples were stored at 4 °C.
For measuring the size (hydrodynamic diameter, dH) of liposomes and their polydispersity index (PDI) the Malvern Zetasizer Nano series Nano-ZS was used. The sample was directly prepared in a polystyrol/polystyrene cuvette from Sarstedt. 1 to 10 µL of sample (depending on sample concentration) was added to PBS for a total volume of 1000 µL and measured in the zetasizer with a size measuring SOP.
For measuring the zeta potential of liposomes the Malvern Zetasizer Nano series Nano-ZS was used. The sample preparation was performed in a reaction tube. 1 to 10 µL of sample (depending on sample concentration) was added to 10 % PBS for a total volume of 1000 µL. The sample was applied to a folded capillary zeta cell and the sample measured with a zeta potential measuring SOP.
For concentrating nucleotides we evaporated the solvent in a SpeedVac SC100. For the decoy DNA primers we used medium heat with a vacuum of below 30 bar for 1 hour or longer until the desired concentration was achieved. The sample was measured with a NanoDrop 2000. For siRNA the vacuum centrifuge was set on low temperature at under 30 bar for 1 hour or longer.
For different experiments knowing the concentration of DNA primers and siRNA was elemental. We used a NanoDrop 2000 for this task. 1 µL of water or 1 µL of the buffer that the nucleic acid was dissolved in was used as blank. The next step was to measure 1 µL of the sample. The measuring device was cleaned before and after each measurement with a non-linting cloth.
In order to evaluate the encapsulation efficiency (EE), we adapted a protocol published by Li & Al-Jamal in 2021.
First, 20 µL of the final liposome solution were loaded with an appropriate amount of loading buffer (in our case 7 µL of
6x Loading Buffer, to ensure that the solution completely drops down and stays in the pocket) onto an 1.8 % Agarose gel stained with
1:10000 ethidium bromide. It was run for 20 min at 85 V in 1x TAE, so that free DNA or siRNA was separated from the liposomes that
had encapsulated DNA or siRNA. Since Liposomes are larger than free nucleotide strands, they stayed within the pocket or moved
significantly slower.
To effectively analyse the amount of nucleotides that was not encapsulated by the liposomes, DNA or siRNA was pipetted into one
pocket as reference. By comparing band intensities with this standard the amount of unencapsulated nucleotides could be measured.
By subtracting and then dividing the obtained value from the initial amount of DNA or siRNA in the given liposome solution, one could
calculate the EE of the liposomes with the following equation.
To test if nucleotides bind to SiLibeads, we incubated a solution of DNA primers in PBS with and without SiLibeads for one day (100 µL of 0.78 mg/mL DNA with 142 mg SiLibeads). We measured the DNA concentration after 0 min, 5 min, 10 min, 30 min, 1 h, 2 h and 1 d with the NanoDrop 2000 and compared the measurements with a sample of PBS.
In order to coat our loaded liposomes with chitosan, we adapted a protocol published by Mady et al. in 2009. 0.3 g chitosan was dissolved in 1 % (w/v) acetic acid. Then, the desired amount of the chitosan solution was added to 10 µL of the liposome solution in a small Eppendorf tube. The mixture was first incubated at room temperature and 400 rpm in a heat block for 2 h. Then, it was incubated overnight at -4°C.
Fe3+: A small crystal of NH4SCN was added into 50 µL of the sample to be tested. Positive control was conducted with a small spatula tip of iron(III)chloride. If the solution contains Fe3+, it will turn dark red (compare with pos. control), and if not, the solution will retain its original colour.
Fe2+: 50 µL of the sample to be tested was first acidified with 10 µL 1M HCl, then a small crystal of K3[Fe(CN)6] was added to the solution. Positive control was conducted with a small spatula tip of iron(II)chloride. If the solution contains Fe2+, it will turn blue (or greenish if there is only a small amount of Fe2+, always compared with pos. control), and if not, the solution will be slightly yellowish due to the colour of K3[Fe(CN)6] itself in an aqueous solution.
The HeLa cells were grown to 80 to 90 % confluency in DMEM supplied with 10 % FCS and 1 % Pen-Strep (Standard DMEM). The cells were trypsinized with 0.025 % trypsin-EDTA and seeded at a density of 2.1 * 10^6 cells in a T75 flask.
The cells were seeded at a density of 3 * 10^5 cells per well in a 6 well-plate in triplicates and incubated for 4 to 6 h at 37 °C in 2 mL standard DMEM. Medium was then replaced with 1.5 mL of Pen-Strep free medium and the cells were then transfected with 6 µg of pEGFP-C1 in 10 µL PEI-max in a total volume of 0.6 mL for 12 h at 37 °C, 5 % CO2. Transfection medium was replaced with 1.5 mL standard DMEM medium before transferring the plate to the IncuCyte. Pictures were then taken every 2 h for 3½ consecutive days. These were then analysed for determination of EGFP expression pattern using R-Studio.
HeLa cells were grown to a confluency of 80 to 90 %, were then harvested with Trypsin-EDTA and seeded at a density of 1.5 * 10^5 cells in 1 mL of standard DMEM. Cells were left 4 to 6 hours to adhere and were then transfected with either 3 µg of pEGFP-C1 or pRK5-HSV UL19 AA816:1148-6xHis using PEI-Max. Transfection media was removed after 12 h and replaced with standard DMEM. After 24 h cells were treated with the liposomal formulations according to Figure 1.
Cells were incubated in the respective treatment solution for transfection with
after 4 ½ hours. The treatment solution was then removed and replaced with 1 mL standard DMEM. Cells were incubated for 2 ½ days to wait for knockdown induction. The experiment was then stopped by Trypsin-EDTA treatment an1 mL of standard DMEM was added to stop the trypsin treatment. The detached cells were transferred into 1.5 mL eppis per well on ice.
A 750 µL aliquot was taken from each sample. This aliquot underwent RNA isolation using the QIAGEN RNeasy Plus Mini Kit. RNA concentration was measured using Nanodrop 2000 and the quality of the isolated RNA was assessed by 2 % agarose-gel electrophoresis. Each sample was used as a triplicate for bulk-RNAseq with each triplicate containing 350 ng total RNA. Bulk-RNAseq was carried out at GeneCore Facility (EMBL Heidelberg).
Three different primer sets were designed using Primer3 for detection of EGFP-RNA and UL19-RNA. Primers were evaluated for proposed annealing temperatures by T-Gradient PCR using 2x Taq Master-Mix (NEB) in a total volume of 25 µL. Plasmid titration was conducted with each primer set to evaluate primer efficiency and Cut-Off values of the Luna Universal One-Step RT-qPCR Kit (NEB) using pEGFP-C1 and pRK5-HSV UL19 AA816:1148-6xHis as template (for plate and reagent setup see Figure 2) in a Roche LightCycler480. Reaction setup was carried out according to the manufacturer's protocol. Standard ranged from 1.31 * 10^10 to 1.31 * 10^2 copies/µL and 5.,37 * 10^9 to 5.37 * 10^1 copies/µL for pEGFP-C1 and pRK5-HSV UL19 AA816:1148-6xHis respectively. To validate selective binding High-Resolution-Melt (HRM) analysis was performed.
| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| BBa_K4344023 | HSV-UL19-fwd-1 | 5’-ACCCAATTTTCCACCAGCAC-3’ | 56.1 | 64.0 | 60.0 |
| BBa_K4344026 | HSV-UL19-rev.-1 | 5’-GCTGATGATGTAAGGCCACG-3’ | 56.1 | 64 | 60 |
| BBa_K4344021 | HSV-UL19-fwd-2 | 5’-AGCATTAGACAGGTGGT-3 | 56.3 | 58.0 | 60.0 |
| BBa_K4344024 | HSV-UL19-rev-2 | 5’-CAAATCTGTCTTGGCGCACT-3’ | 56.0 | 58.0 | 60.0 |
| BBa_K4344022 | HSV-UL19-fwd-3 | 5’-TCAGGTTCGATCGGGTGTAC-3’ | 56.4 | 64.0 | 60.0 |
| BBa_K4344025 | HSV-UL19-rev-3 | 5’-TGTGCGAGAACCTGAAGAGT-3’ | 56.1 | 64.0 | 60.0 |
| BBa_K4344009 | EGFP-fwd-1 | 5’-GGTGAACTTCAAGATCCGCC-3’ | 56.1 | 64.0 | 60.0 |
| BBa_K4344012 | EGFP-rev-1 | 5’-CTTGTACAGCTCGTCCATGC-3’ | 56.0 | 64.0 | 60.0 |
| BBa_K4344010 | EGFP-fwd-2 | 5’-GACGACGGCAACTACAAGAC-3’ | 55.8 | 61.0 | 60.0 |
| BBa_K4344013 | EGFP-rev-2 | 5’-GGCGGATCTTGAAGTTCAC-3’ | 53.6 | 61.0 | 60.0 |
| BBa_K4344011 | EGFP-fwd-3 | 5’-TAAACGGCCACAAGTTCAGC-3’ | 55.9 | 64.0 | 60.0 |
| BBa_K4344014 | EGFP-rev-3 | 5’-AAGTCGTGCTGCTTCATGTG-3’ | 55.9 | 64.0 | 60.0 |
Knockdown efficiency was evaluated by RT-qPCR using the Luna Universal One-Step RT-qPCR Kit (NEB). In case of UL19 primer set 1 was used. GAPDH served as a housekeeping gene reference. Samples used for bulk RNA-Seq were pooled per condition and treatment and purified with Monarch RNA Extraction Kit. RT-qPCR was carried out as described before (compare Figure 2 B &D). A total of 50 ng RNA was used per reaction. Samples were analysed in duplicates. Figure 3 visualises the pipetting scheme.
| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| BBa_K4344016 | GAPDH-fwd. | 5’-GTCTCCTCTGACTTCAACAGCG-3’ | 57.4 | 65.0 | 60.0 |
| BBa_K4344017 | GAPDH-rev. | 5’-ACCACCCTGTTGCTGTAGCCAA-3’ | 61.3 | 65.0 | 60.0 |
The remaining 750 µL of the triplicates were pooled and the cells were pelleted at 16000 rpm at 4°C for 5 min. Supernatant was removed and replaced by a protein lysis buffer. The mixture was well-vortexed. Samples were then kept on ice to precipitate excess SDS. The precipitate was spun down at 16000 rpm at 4°C for 15 min. The supernatant was transferred to a 1.5 mL eppi. For measurement of protein concentration a Bradford Assay was conducted as described earlier. 40 µg of total protein for each condition were loaded onto a SDS-Page. 5 µL prestained protein ladder was loaded as a size standard. The gel was run at 110 V for 1½ h on ice. The proteins were extracted from the gel by semi dry Western Blotting on a nitrocellulose membrane at 15 V for 60 min. The blot membrane was blocked with 5 % milk powder in TBS-T for 1 h. Afterwards blot was incubated with primary antibody Anti-His-tag mouse monoclonal antibody (1:10000, Proteintech) in case of pRK5-HSV UL19 AA816:1148-6xHis or anti-GFP-tag mouse monoclonal antibody (1:20000) in blocking solution supplied with 10 mM sodium azide at 4°C overnight. Secondary antibody goat anti-mouse HRP conjugated antibody(1:5000, Proteintech) in blocking solution supplied with 10 mM sodium azide at 4°C for 2 h. The membrane was washed five times for 3 min to remove unselective bound antibodies. After that the membrane was stained with DAB to detect UL19 fragments or EGFP. α-tubulin served as a housekeeping gene for relative quantification, anti-tubulin mouse monoclonal antibody (1:20000, Proteintech) was used. The images were analysed using a ChemiDoc XRS+ (BioRad Laboratories).