Introduction
Initial translation rates were measured by transient expression of DNA plasmids containing a modified 5’ UTR with iron response elements verified with IRBPs (Iron Responsive Binding Proteins) endogenous to single-cell CHO. The IRE sequences selected are of the wildtype one found in the human ferritin 5’ UTR that binds to both IRBP 1 and IRBP2 proteins (also referred to as ACO1 and ACO2 respectively). Other IRE structures used were specifically mutated to change the non-conserved “loop” region of the stem-loop structure resulting in IRE structures that only bind to IRBP1, IRBP2, or neither of them as demonstrated in Menotti et. al. 1998. Additionally, Menotti’s team conducted a western blot analysis on a native-PAGE gel to confirm that endogenous-CHO IRBP1 and IRBP2 were in complex with the designed mRNA IRE stem-loops and that the proteins were expressed natively.
The RNA design is as follows. A putative 5’ UTR Moderna vaccine sequence assembly was collected from Dae-Eun et. al. 2021 and was used to demonstrate the ability to insert the IRE into another novel 5’ UTR and still retain translational regulation ability. The mRNA design encodes for two proteins that are separated during translation via a P2A cleavage site. A VSV-G protein with a plasma membrane localization signal with HA tag was encoded upstream of the P2A cleavage site. This protein will accumulate in the membrane. After fluorescence microscopy for translation data collection, a western blot with antibodies against the HA was conducted to assess the total translation of mRNA.
Downstream of the P2A cleavage site, a YFP with a fused destabilized domain was encoded to measure the translation rate over a period of 12 hours. The ecDHFR (dihydrofolate reductase region) destabilized YFP is marked for immediate protease degradation and thus no fluorescence can be observed. The TMP (trimethoprim) ligand addition binds to the ecDHFR region and blocks degradation. This allows us to induce the YFP promoter at different times to produce an accumulating rate of fluorescence to be monitored.
The 3’ Region of the mRNA design contains a beta-globin sequence as described in Adibzadeh et al. 2019. To prolong mRNA stability in the cell by reducing 3’ to 5’ exonuclease activity. All these inserts were assembled by Gibson Assembly and integrated into the pcDNA3.1(+) mammalian expression vector synthesized by Gen Script.
CHO DG44 cells were cultured in a complete medium containing DMEM, FBS, Penicillin, and HT. Cells were plated in T75 flasks and grown in a CO2 incubator at 37℃. Media is changed every other day and cells must be passed every three days to not reach maximum confluence.
Once cells had grown to full confluence, by a 1:5 dilution we moved our cells into a 96 well plate imaging plate for transfection. Our plasmid, along with Lipofectamine 3000, was incubated overnight in a CO2 incubator at 37℃ to promote ample integration of our plasmids. TOP-H2B-YFP-DD, IRES-H2B-YFP-DD, and UnReg-H2B-YFP-DD. Provided by Dr. Kyuho Han at Stanford University. After incubation, the 96 well plate was brought over to our wide-field epifluorescence microscope. A Nikon Eclipse Ti with NIS Elements control software, Andor Zyla 5.5 camera, Lumencor SOLA light source, and OKO Labs stage-top incubator.
Movies were taken of each of our 60 transfected wells. Each movie contained a group of 175 images taken in 6-minute intervals. TMP was added at time point 58. Post treatment, we have twelve hours of fluorescence documented for our further processing
After the microscope had finished its 175th-time step, we analyzed the cell movies using NIS Element Viewer. Allocating different channels to each of the different reporters allowed us to see our nuclear staining before drug treatment, and mCardinal and YFP fluorescence after treatment. The nd2 metadata file received from the epifluorescence microscope is then run through two MatLab scripts to filter through the data and create plots. First, celltracer, to track which cells were successfully transfected, and then plot_by to create heatmaps and translation over time graphs.
Addgene link to the assembled plasmid. - https://www.addgene.org/178204/
5’ --IRE(Ferritin)--Moderna 5’UTR--VSVG Signal Peptide--VSVG(excretion protein)--P2A(Self-cleaving peptide)--H2B(nuclear localization signal)--YFP w/ Destabilization domain--100 to 200bp 3’ Beta-globin UTR--3’
Wildtype IRE moderna 5’ UTR VSV G/ddYFP
GATCCTGCTTCAACAGTGCTTGGACGGACGGATCTTGGGAAATAAGA GAGAAAGATCCTGCTTCAACAGTGCTTGGACGGACGGATCTTAGAAG AGTAAGAAGAAATATAAGACCCCGGCGCCGCCACCATGAAGTGCCTT TTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCAT AGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTA ATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGAC TTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGC TATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTA CTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATC CGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACA AACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTT GTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTG ACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGA TTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTG TCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTA TGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGA CGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTA ACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATAC TGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGAT GGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAG AAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGT CTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGA AACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATC TCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACC ATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGT CGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTG GAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAA GACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATA TAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATC TTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAA GACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGA TACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCA GTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTA ATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCAT TAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGA TGAACCGACTTGGAAAGTAAGGAAGCGGAGCTACTAACTTCAGCCTG CTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGCCAGA GCCAGCGAAGTCTGCTCCCGCCCCGAAAAAGGGCTCCAAGAAGGCGG TGACTAAGGCGCAGAAGAAAGGCGGCAAGAAGCGCAAGCGCAGCCGC AAGGAGAGCTATTCCATCTATGTGTACAAGGTTCTGAAGCAGGTCCA CCCTGACACCGGCATTTCGTCCAAGGCCATGGGCATCATGAATTCGT TTGTGAACGACATTTTCGAGCGCATCGCAGGTGAGGCTTCCCGCCTG GCGCATTACAACAAGCGCTCGACCATCACCTCCAGGGAGATCCAGAC GGCCGTGCGCCTGCTGCTGCCTGGGGAGTTGGCCAAGCACGCCGTGT CCGAGGGTACTAAGGCCATCACCAAGTACACCAGCGCTAAGGATCCA GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCG AGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATC TGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGA AGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG GAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGA AGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCA GAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCC TGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG CTGTACAAGCTCGAGATCAGTCTGATTGCGGCGTTAGCGGTAGATCA CGTTATCGGCATGGAAACCGTCATGCCGTGGAACCTGCCTGCCGATC TCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGC CGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAA TATTATCCTCAGCAGTCAACCGAGTACGGACGATCGCGTAACGTGGG TGAAGTCGGTGGATGAAGCCATCGCGGCGTGTGGTGACGTACCAGAA ATCATGGTTATTGGCGGCGGTCGCGTTTATGAACAGTTCTTGCCAAA AGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCG ACACCCATTTCCCGGATTACGAGCCGGATGACTGGGAATCGGTATTC AGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTT TGAGATTCTGGAGCGGCGATGAATGCGCGGCCGCACTCCTCAGGTGC AGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCT CACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGA CATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAAT TTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTC GGAAGGACATATGGG
IRP1 IRE in moderna 5’ with VSV G/ddYFP
GATCCTGCTTCAATAGTACTTGGACGGACGGATCTTGGGAAATAAGAG AGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACCATG AAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAG TTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTT CCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCAT AATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCAC AAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTC ACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCC ATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAA CAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGT TGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTG ACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGAT TCTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCA TGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAA AGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAG GCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCC CATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAG CCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTC AGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGG ATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTC CAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAA CCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGA CCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGG TCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACT GGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGA CCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGT TGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATC CTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTAT TTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAG GTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCA TAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATC TTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACA TAGAGATGAACCGACTTGGAAAGTAAGGAAGCGGAGCTACTAACTTCA GCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGC CAGAGCCAGCGAAGTCTGCTCCCGCCCCGAAAAAGGGCTCCAAGAAGG CGGTGACTAAGGCGCAGAAGAAAGGCGGCAAGAAGCGCAAGCGCAGCC GCAAGGAGAGCTATTCCATCTATGTGTACAAGGTTCTGAAGCAGGTCC ACCCTGACACCGGCATTTCGTCCAAGGCCATGGGCATCATGAATTCGT TTGTGAACGACATTTTCGAGCGCATCGCAGGTGAGGCTTCCCGCCTGG CGCATTACAACAAGCGCTCGACCATCACCTCCAGGGAGATCCAGACGG CCGTGCGCCTGCTGCTGCCTGGGGAGTTGGCCAAGCACGCCGTGTCCG ATGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCC GGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC ACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG GAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCC GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAG TACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAG AACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGAC GGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCC CTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAG TTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG CTCGAGATCAGTCTGATTGCGGCGTTAGCGGTAGATCACGTTATCGGC ATGGAAACCGTCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTT AAACGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGG GAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGC AGTCAACCGAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGAT GAAGCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTTATTGGC GGCGGTCGCGTTTATGAACAGTTCTTGCCAAAAGCGCAAAAACTGTAT CTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGAT TACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCT GATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGA TGAATGCGCGGCCGCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTG GTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTT TTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCAT CTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGT TGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGG
IRP2 IRE in moderna 5’ with VSV G/ddYFP
GATCCTGCTTCAACCGAGCTTGGACGGACGGATCTTGGGAAATAAGAG AGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACCATG AAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAG TTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTT CCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCAT AATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCAC AAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTC ACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCC ATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAA CAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGT TGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTG ACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGAT TCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTC CATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGT GATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGA GAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTAC TTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAG CATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGAT AAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCA AGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAG GACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGC AAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTT GCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGT ACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCT CCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAA AGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGA CCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATAC ATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAG GCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTT CCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAAT CCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATT GCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTT CTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAA AGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTAAGGA AGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCTATGCCAGAGCCAGCGAAGTCTGCTCCCGCCCCG AAAAAGGGCTCCAAGAAGGCGGTGACTAAGGCGCAGAAGAAAGGCGGC AAGAAGCGCAAGCGCAGCCGCAAGGAGAGCTATTCCATCTATGTGTAC AAGGTTCTGAAGCAGGTCCACCCTGACACCGGCATTTCGTCCAAGGCC ATGGGCATCATGAATTCGTTTGTGAACGACATTTTCGAGCGCATCGCA GGTGAGGCTTCCCGCCTGGCGCATTACAACAAGCGCTCGACCATCACC TCCAGGGAGATCCAGACGGCCGTGCGCCTGCTGCTGCCTGGGGAGTTG GCCAAGCACGCCGTGTCCGAGGGTACTAAGGCCATCACCAAGTACACC AGCGCTAAGGATCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTG GTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTC AGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACC CTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCC GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGC TACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAG ACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATC GAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCC ATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTAC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG CTGTACAAGCTCGAGATCAGTCTGATTGCGGCGTTAGCGGTAGATCAC GTTATCGGCATGGAAACCGTCATGCCGTGGAACCTGCCTGCCGATCTC GCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGCCGC CATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATT ATCCTCAGCAGTCAACCGAGTACGGACGATCGCGTAACGTGGGTGAAG TCGGTGGATGAAGCCATGCGGCGTGTGGTGACGTACCAGAAATCATGG TTATTGGCGGCGGTCGCGTTTATGAACAGTTCTTGCCAAAAGCGCAAA AACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATT TCCCGGATTACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCC ACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGG AGCGGCGATGAATGCGCGGCCGCACTCCTCAGGTGCAGGCTGCCTATC AGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACT GAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCC TTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAA TAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGG
Control IRE (no IRBP binding) in moderna 5’ with VSV G/ddYFP
Control IRE: GATCCTGCTTCAAGCTCCGTTGGACGGACGGATCTT GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATGATCCTGCTTCAAG CTCCGTTGGACGGACGGATCTTATAAGACCCCGGCGCCGCCACCATGAA GTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTC ACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTT CTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGA CTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCT ATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTT GTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATC CTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAA CAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATG CAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCA TGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATC AACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAA CCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCAT TTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTG GGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTG GAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACT CCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCA GCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTC AGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGA TTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCA ATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCG GTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAG ATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGA ATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCAC CATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTC AGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCC GATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTC AAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGA TACTGGGCTATCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTA GTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCAT TGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTA AAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGAC TTGGAAAGTAAGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGC TGGAGACGTGGAGGAGAACCCTGGACCTATGCCAGAGCCAGCGAAGTCT GCTCCCGCCCCGAAAAAGGGCTCCAAGAAGGCGGTGACTAAGGCGCAGA AGAAAGGCGGCAAGAAGCGCAAGCGCAGCCGCAAGGAGAGCTATTCCAT CTATGTGTACAAGGTTCTGAAGCAGGTCCACCCTGACACCGGCATTTCG TCCAAGGCCATGGGCATCATGAATTCGTTTGTGAACGACATTTTCGAGC GCATCGCAGGTGAGGCTTCCCGCCTGGCGCATTACAACAAGCGCTCGAC CATCACCTCCAGGGAGATCCAGACGGCCGTGCGCCTGCTGCTGCCTGGG GAGTTGGCCAAGCACGCCGTGTCCGAGGGTACTAAGGCCATCACCAAGT ACACCAGCGCTAAGGATCCAGTGAGCAAGGGCGAGGAGCTGTTCACCGG GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAG TTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGA CCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCAC CCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCC GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCT ACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCA GAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGAC GGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGC CCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAG TTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGC TCGAGATCAGTCTGATTGCGGCGTTAGCGGTAGATCACGTTATCGGCAT GGAAACCGTCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAAA CGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGGGAAT CAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCA ACCGAGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAAGCC ATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTTATTGGCGGCGGTC GCGTTTATGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGCA TATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCCG GATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGA ACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGATGAATGCGCGG CCGCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTG GCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTA ATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGT CTCTCACTCGGAAGGACATATGGG
No IRE control, Pfizer 5’ with VSV G/ddYFP:
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCG CCGCCACCATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGT GAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGG AAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAA ATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAA GAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAA TGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACAC ATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCAT TGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAA AGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGG TGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGA TTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTC CATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTG ATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGA GCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTT GCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATT GGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGA TCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATC TCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTG AGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAG AGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAA AACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAAT ACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTC AAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGG GATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTC TGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGG TATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAA CATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTT TATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGA AGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATC ATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATC TTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACAT AGAGATGAACCGACTTGGAAAGTAAGGAAGCGGAGCTACTAACTTCAGC CTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGCCAG AGCCAGCGAAGTCTGCTCCCGCCCCGAAAAAGGGCTCCAAGAAGGCGGT GACTAAGGCGCAGAAGAAAGGCGGCAAGAAGCGCAAGCGCAGCCGCAAG GAGAGCTATTCCATCTATGTGTACAAGGTTCTGAAGCAGGTCCACCCTG ACACCGGCATTTCGTCCAAGGCCATGGGCATCATGAATTCGTTTGTGAA CGACATTTTCGAGCGCATCGCAGGTGAGGCTTCCCGCCTGGCGCATTAC AACAAGCGCTCGACCATCACCTCCAGGGAGATCCAGACGGCCGTGCGCC TGCTGCTGCCTGGGGAGTTGGCCAAGCACGCCGTGTCCGAGGGTACTAA GGCCATCACCAAGTACACCAGCGCTAAGGATCCAGTGAGCAAGGGCGAG GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACG TAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCC GTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCT TCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGC CATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGAC GGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGG TGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACAT CCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCC ACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAA CACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTG AGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACA TGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGA CGAGCTGTACAAGCTCGAGATCAGTCTGATTGCGGCGTTAGCGGTAGAT CACGTTATCGGCATGGAAACCGTCATGCCGTGGAACCTGCCTGCCGATC TCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGCCG CCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATT ATCCTCAGCAGTCAACCGAGTACGGACGATCGCGTAACGTGGGTGAAGT CGGTGGATGAAGCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGT TATTGGCGGCGGTCGCGTTTATGAACAGTTCTTGCCAAAAGCGCAAAAA CTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCC CGGATTACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGA TGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGG CGATGAATGCGCGGCCGCACTCCTCAGGTGCAGGCTGCCTATCAGAAGG TGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCT TTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCAT CTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTT GGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGG