Hype s imula o y N⁶-me hyladenine (m6A) in esidual SV40
plasmid DNA in mRNA accines.
Ke in McKe nan
Medicinal Genomics, Be e ly MA
Abs ac
Many mRNA accine p oduc ion pipelines ely on Esche ichia coli o eplica e plasmid DNA
empla es used in he in i o ansc ip ion o modi ied RNA. Howe e , E. coli DNA me hyla ion
pa e ns di e subs an ially om hose o humans. In E. coli, DNA me hyla ion is p ima ily
media ed by DNA adenine me hyl ans e ase (Dam), which in oduces N⁶-me hyladenine (m6A)
wi hin GATC mo i s, whe eas human me hyla ion occu s p edominan ly a cy osines in CpG
dinucleo ides. Some E. coli s ains also exp ess Dcm me hyl ans e ase, which me hyla es
CCWGG sequences (CC[A/T]GG), u he dis inguishing bac e ial om mammalian epigene ic
ma ks.
Cy osolic DNA ha lacks CpG me hyla ion can po en ly ac i a e Toll-like ecep o 9 (TLR9),
while m6A-modi ied DNA has been shown o s imula e he cGAS–STING pa hway, leading o
he induc ion o ype I in e e ons and o he in lamma o y media o s.
Because he P ize mRNA accine plasmids a e p opaga ed in E. coli, and esidual plasmid DNA
has been de ec ed in inished accine ma e ial, i is likely ha his DNA bea s bac e ial- ype
me hyla ion pa e ns ha could be immunos imula o y h ough TLR9 and cGAS–STING
signaling. To in es iga e his possibili y, we applied Ox o d Nanopo e sequencing o examine
he me hyla ion s a us o plasmid DNA p esen in P ize lo FL8095.
Figu e 1. Red do s e lec me hyla ion o N⁶-me hyladenine in he palind omic sequence GATC
(image sou ce- h ps://2009.igem.o g/Team:Impe ial_College_London/M3/DamMe hyla ion)
Me hods
We pu i ied 600µl o P ize lo FL8095 using 6µl o 10% T i on X. A e a quick o ex samples
we e hea ed a 95°C o 2 minu es and hen spun a 15,000 RPM o 5 minu es. Samples a e
placed on ice o ha den he op oil laye and 2.5µl o RNaseA (Mona ch RNaseA -NEB) is added
o he bo om laye and incuba e a 37°C o 10 minu e.
Pu i ica ion o he small DNA was accomplished wi h a modi ied Ampu e p o ocol using 600µl
o Ampu e, 600µl o 100% Isop opanol and 12µl 1M MgCl2. Samples a e mixed and allowed o
bead bind o 10 minu es. A magne is used o sepa a e he beads and hey a e washed wice wi h
1ml 70% E OH. DNA is elu ed in 30µl o ddH20.
Figu e 2. P ize /BioN ech FL8095 sample a e T i onX, Hea and Cen i uga ion.
DNA Sequencing lib a ies we e cons uc ed wi h Ox o d Nanopo es ONT liga ion sequencing
assay V14. Two modi ica ions we e made o he de aul p o ocol o be e cap u e small DNA.
The Ampu e s ep a e he End Repai s ep was inc eased om 60µl o 90µl. The Ampu e s ep
a e he Liga ion s ep was inc eased om 40µl o 100µl.
These lib a ies we e loaded on o an Ox o d Nanopo e MinION Mk1D using hei R10.4.1 low
cells. Sequencing eads we e base called wi h he Do ado base calle using he
[email p o ec ed]_6mA@ 1 model. Minimap2 was used o align he eads
o NCBI e e ence OR134577.1.
Assessmen o Eam1104I linea iza ion:
Ox o d Nanopo e eads we e aligned o he P ize /BioNTech plasmid e e ence (GenBank
accession OR134577.1) o assess he comple eness o linea iza ion a he Eam1104I si e.
To accu a ely de ec eads spanning he linea iza ion junc ion, he e e ence was conca ena ed
(i.e., duplica ed end- o-end) so ha alignmen s could occu ac oss he Eam1104I cu si e.
Mapping eads o he e minal ends o a single-copy e e ence would o he wise esul in minimal
alignmen s because mos ead mappe s do no handle ci cula e e ence con inui y.
Reads a e sing he Eam1104I junc ion we e hen isualized in IGV o con i m incomple e
linea iza ion e en s.
Resul s
591,304 eads wi h p ima y alignmen s o he P ize e e ence (OR134577.1) we e gene a ed.
DAM me hyla ion can be seen on bo h s ands o he GATC palind omic sequence. Top s and is
G ey and bo om s and is G een. Dam me hyla ion can be obse ed (Figu e 4-7) and a ies
ac oss he plasmid (Figu e 8).
Figu e 3. Aligned ONT Read leng h dis ibu ions. Ox o d Nanopo es canno h ead ci cula
DNA h ough he po es and canno be measu ed wi h hese me hods.
Figu e 4. IGV display o Dam me hyla ion (GATC) in Plasmid DNA sequence om P ize lo
FL8095
Figu e 5. Tandem GATC si es hea ily me hyla ed
Figu e 6. This s ain o E.coli does no me hyla e he SV40 p omo e s.
Figu e 7. The spike ORF is densely me hyla ed.
Figu e 8. Me hyla ion Hea map ac oss he plasmid demons a e hypome hyla ion o SV40
componen s while hype me hyla ion o Neo/Kan and Spike.
Figu e 9. Ox o d Nanopo e sequencing wo k low illus a ing he Eam1104I diges ion s a egy
o linea iza ion o he P ize /BioNTech plasmid p io o RNA ansc ip ion
Figu e 10. IGV isualiza ion o Ox o d Nanopo e eads spanning he Eam1104I cu si e, showing
ead- h ough ac oss he expec ed junc ion and incomple e plasmid linea iza ion.
E idence o Incomple e Linea iza ion a he Eam1104I Si e
Ox o d Nanopo e sequencing e ealed mul iple eads a e sing he Eam1104I linea iza ion si e
used in he p oduc ion o P ize /BioNTech’s mRNA accine plasmids. This inding indica es
incomple e diges ion du ing he in i o linea iza ion s ep, sugges ing ha a small ac ion o
plasmid molecules may ha e emained ci cula ized (Figu e 9). Reads spanning he expec ed cu
junc ion (Figu e 10) demons a e con inui y ac oss bo h clea age posi ions, consis en wi h
esidual in ac plasmids pe sis ing in he inal o mula ion.
The p esence o ci cula plasmids is no expec ed o a ise pos -diges ion, as hos ligases a e
absen du ing he in i o ansc ip ion p epa a ion. Howe e , once injec ed in o mammalian
issue, hos ligases could po en ially e-ci cula ize linea plasmid agmen s ha e ain
complemen a y 5′ o e hangs. This aises a heo e ical conce n ha a e, eplica ion-compe en
plasmids could e o m in i o.
The longes con inuous ead spanning he Eam1104I si e exceeds 3,400 bases, con i ming ha
long esidual agmen s su i e pu i ica ion and a e se he nominal clea age si e. Because
Ox o d Nanopo e pla o ms canno p ocess ci cula DNA, he exac abundance o ull-leng h
ci cula plasmids canno be di ec ly measu ed wi hou addi ional enzyma ic linea iza ion o
esidual DNA. Ne e heless, hese indings demons a e ha incomple e diges ion and low-le el
pe sis ence o ci cula o ms canno be excluded in he accine DNA empla e.
Discussion
These da a i mly es ablish ha P ize /BioN ech plasmid manu ac u ing did no u ilize Dam
knock ou E.coli s ains. DNA con aining N⁶-me hyladenine (m6A) is inc easingly ecognized as
an immunos imula o y signal when de ec ed by he hos inna e immune sys em. Recen s udies
ha e shown ha m6A-modi ied DNA can ac as a po en ac i a o o he cGAS–STING
pa hway, enhancing in e e on esponses and ampli ying downs eam in lamma o y signaling
(Balza olo e al., 2021). The cGAS–STING sys em i sel is he p ima y cy osolic DNA senso ,
esponsible o de ec ing o eign DNA and igge ing ype I in e e on p oduc ion (Li & Chen,
2016).
P omo e and enhance egions a e o en sensi i e o DNA me hyla ion, and classical s udies
wi h cy osine me hyla ion ha e shown ha me hyla ion wi hin he SV40 egula o y egion can
educe p omo e -d i en gene exp ession (B yans e al., 1992). Al hough hose s udies did no
examine adenine me hyla ion, hey p o ide p eceden ha me hyl g oups wi hin egula o y
sequences can in luence ch oma in accessibili y o p omo e ac i i y. In he con ex o m6A
(Dam me hyla ion), i emains plausible ha me hyla ion a GATC si es in he SV40 egion could
al e ansc ip ion ac o binding o ch oma in s a e, al hough di ec e idence in his sys em is
lacking.
Recen single-molecule ch oma in p o iling o plasmids demons a ed ha p omo e iden i y
s ongly in luences nuclea impo and ch oma iniza ion (Mallo y e al., 2025). These indings
sugges ha epigene ic and s uc u al ea u es o plasmids oge he go e n ansc ip ional
accessibili y.
The da a he e indica e ha while he plasmid backbone is b oadly me hyla ed a GATC si es, he
SV40 p omo e s and enhance s emain unme hyla ed in his E. coli s ain. This selec i e lack o
me hyla ion sugges s ha he bac e ial hos does no a ge hese egula o y elemen s o m6A
modi ica ion, he eby po en ially p ese ing hei ull ansc ip ional ac i i y. A he same ime,
pe asi e m6A me hyla ion elsewhe e in he plasmid may ac as a hype -s imula o y signal o
he inna e immune sys em ia cGAS–STING.
Beyond he SV40 egula o y egion i sel , bac e ial me hyla ion s a e has been shown o
in luence he biological pe o mance o plasmid DNA in mammalian sys ems (Ca nes e al.,
2010). Vi o e al. (2011) epo ed ha dcm⁻ plasmids, which lack cy osine me hyla ion a
CCWGG mo i s, exhibi ed enhanced ansgene exp ession in human cells bu pa adoxically
educed immunogenici y. This highligh s ha plasmid me hyla ion pa e ns—bo h a adenine
(Dam) and cy osine (Dcm) si es—can ha e measu able downs eam e ec s on exp ession and
inna e immune ac i a ion. Thei obse a ion ha plasmid me hyla ion s a us should be
s anda dized ea ly in de elopmen unde sco es he ele ance o epigene ic s a e o plasmid-based
accine and gene he apy ec o s.
Impo an ly, ch onic ac i a ion o cGAS–STING signaling has been linked o oncogenic
p ocesses. Kwon e al. (2020) e iew e idence showing ha while acu e pa hway ac i a ion is
p o ec i e—p omo ing an i i al and an i umo immuni y—pe sis en cGAS–STING s imula ion
can d i e noncanonical NF-κB signaling, in lamma ion, genomic ins abili y, and umo
p og ession. Sus ained cy osolic DNA s ess blun s ype I in e e on ou pu and shi s he
ansc ip ional p og am owa d p o-su i al and immunosupp essi e signaling. In his con ex ,
esidual plasmid DNA bea ing m6A ma ks could, i pe sis en ly sensed, p omo e maladap i e
in lamma ion and possibly con ibu e o oncogenic isk h ough ch onic STING engagemen .
I has p e iously been epo ed ha di e en ial gene exp ession analyses be ween accina ed and
un accina ed coho s e ealed al e ed exp ession o genes in he cGAS-STING pa hway (Lee e
al., 2022; McKe nan, 2025). In hese RNA-seq da ase s, eads co esponding o plasmid DNA
sequences om he accine we e de ec ed in he Sequence Read A chi e (SRA). Al hough
Illumina sequencing lacks me hyla ion de ec ion capabili y, he p esence o hese plasmid-
de i ed eads suppo s p io obse a ions o esidual DNA in accina ed indi iduals.
Impo an ly, mos RNA-seq p o ocols employ DNase I o Ac inomycin D o supp ess DNA
con amina ion, ye accine-de i ed plasmid DNA signals pe sis e en unde hose condi ions—
consis en wi h he po en ial o s able, immunos imula o y DNA capable o engaging he cGAS–
STING axis.
Taken oge he , hese indings sugges a dual e ec : enhanced immunos imula o y capaci y om
widesp ead m6A modi ica ion, coupled wi h po en ially e ained high ansc ip ional ac i i y o
SV40 egula o y elemen s due o hei unme hyla ed s a us. This in e play may ha e impo an
implica ions o he design o plasmid backbones in mammalian exp ession sys ems, whe e bo h
immune ecogni ion and p omo e s eng h mus be ca e ully balanced.
