BNT162b2
3.2.S.1.2 Structure [Omicron (B.1.1.529) Variant]
3.2.S.1.2. STRUCTURE, OMICRON (B.1.1.529) VARIANT
The active principle in each Omicron (B.1.1.529) variant drug substance (DS) is a single-
stranded, 5'-capped mRNA that is translated into the respective protein (the encoded
antigen). Figure 3.2.S.1.2-1 illustrates the general structure of the antigen-encoding RNA,
which is determined by the respective nucleotide sequence of the DNA used as template for
in vitro RNA transcription. In addition to the codon-optimized sequence encoding the
antigen, the RNA contains common structural elements optimized for mediating high RNA
stability and translational efficiency (5'-cap, 5'-UTR, 3'-UTR, poly(A) - tail; see below).
Furthermore, an intrinsic signal peptide (sec) is part of the antigen-encoding regions and is
translated as N-terminal tag.
Figure 3.2.S.1.2-1.
General structure of the Omicron (B.1.1.529) variant RNA
Schematic illustration of the general structure of the Omicron (B.1.1.529) drug substance with 5'-cap, 5'- and
3'-untranslated regions (hAg-Kozak and FI element, respectively), coding sequence for variant of concern and
intrinsic signal peptide (sec) as well as poly(A)-tail (A30L70). Individual elements are not drawn to scale
compared to their respective sequence lengths.
mRNA cap
A cap1 structure m 7,3’-O
2’-O
2
Gppp(m1
)ApG is utilized as specific capping structure at the 5′-
end of the RNA drug substance (Figure 3.2.S.1.2-2).
Figure 3.2.S.1.2-2.
5′-cap analog (m27,3’-OGppp(m12’-O)ApG) for production of RNA
containing a cap1 structure
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BNT162b2
3.2.S.1.2 Structure [Omicron (B.1.1.529) Variant]
The cap1 structure (i.e., containing a 2′-O-methyl group on the penultimate nucleoside of the
5′-end of the RNA chain) is incorporated into the RNA drug substance by using a respective
cap analog during in vitro transcription. For RNAs with modified uridine nucleotides, the
cap1 structure is superior to other cap structures, since cap1 is not recognized by cellular
factors such as IFIT11 and, thus, cap1-dependent translation is not inhibited by competition
with eukaryotic translation initiation factor 4E2. In the context of IFIT1 expression, mRNAs
with a cap1 structure give higher protein expression.
In addition, use of the cap1 structure leads to low amounts of uncapped transcripts3. In
general, the T7 Polymerase prefers a guanosine as priming nucleoside with the highest
transcription efficiencies as compared to other starting nucleosides4. Capping structures with
a guanosine moiety compete with GTP for incorporation in the mRNA resulting in uncapped
transcripts. The m 7,3’-O
2’-O
2
Gppp(m1
)ApG cap analog rescues transcription efficiency from
templates starting with adenosines, because the ApG moiety of cap1 allows transcription
initiation at the second position, a guanosine, thereby giving mainly capped mRNAs.
Modified Uridine
The RNA does not contain any uridines; instead of uridine the modified
N1-methylpseudouridine is used in RNA synthesis. Several reports have demonstrated that
such a substitution often strongly enhances translation of in vitro transcribed mRNA
sequences by reducing its immunogenicity5,6,7. Accordingly, the drug substance is
synthesized in the presence of N1-methylpseudouridine triphosphate (m1ΨTP) instead of
uridine triphosphate (UTP).

1 Habjan M, Hubel P, Lacerda L, et al. Sequestration by IFIT1 Impairs Translation of 2′O-unmethylated Capped
RNA. 2013. PLOS Pathog;9(10):e1003663
2 Diamond MS. IFIT1: A dual sensor and effector molecule that detects non-2’-O methylated viral RNA and
inhibits its translation. 2014. Cytokine Growth Factor Rev;25(5):543-50.
3 Trilink Patent auf CC413 cap. Accessed at
https://patentimages.storage.googleapis.com/4c/83/15/99418d175a3be2/WO2017053297A1.pdf
4 Kuzmine I, Gottlieb PA, Martin CT. Binding of the priming nucleotide in the initiation of transcription by T7
RNA polymerase. 2003. J Biol Chem;278(5):2819-23.
5 Kariko K, Muramatsu H, Welsh FA, et al. Incorporation of pseudouridine into mRNA yields superior
nonimmunogenic vector with increased translational capacity and biological stability. 2008. Mol
Ther;16(11):1833-40.
6 Andries O, Mc Cafferty S, De Smedt SC, et al. N(1)-methylpseudouridine-incorporated mRNA outperforms
pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in
mammalian cell lines and mice. 2015. J Control Release; 217:337-44.
7 Richner JM, Himansu S, Dowd KA, et al. Modified mRNA Vaccines Protect against Zika Virus Infection.
2017. Cell;168(6):1114-25.e10
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3.2.S.1.2 Structure [Omicron (B.1.1.529) Variant]
RNA sequence
The general sequence elements of the Omicron (B.1.1.529) variant drug substance, as
depicted in Figure 3.2.S.1.2-1, are given below. The full sequence is given in
Figure 3.2.S.1.2-3.
hAg-Kozak (nucleotides 2 to 54): 5'-UTR sequence of the human alpha-globin mRNA with
an optimized ʻKozak sequenceʼ to increase translational efficiency8.
Sec (nucleotides 55 to 102): Sec corresponds to the intrinsic S1S2 protein signal peptide
(sec), which guides translocation of the nascent polypeptide chain into the endoplasmic
reticulum.
S protein omicron (nucleotides 103 to 3870): Codon-optimized sequences encoding the
respective antigen of SARS-CoV-2 protein has following point mutations/deletions
(reference for numbering Genbank ID QHD43416.1): A67V, ΔHV69-70, T95I, G142D,
ΔVYY143-145, ΔN211, L212I, R214_D215insEPE, G339D, S371L, S373P, S375F, K417N,
N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,
D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, KV986-
7PP.
FI element (nucleotides 3871 to 4165): The 3′-UTR is a combination of two sequence
elements derived from the “amino terminal enhancer of split” (AES) mRNA (called F) and
the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an
ex vivo selection process for sequences that confer RNA stability and augment total protein
expression9.
A30L70 (nucleotides 4166 to 4275): A poly(A)-tail measuring 110 nucleotides in length,
consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence
and another 70 adenosine residues designed to enhance RNA stability and translational
efficiency in dendritic cells10.

8 Kozak M. An analysis of 5’-noncoding sequences from 699 vertebrate messenger RNAs. 1987. Nucleic Acids
Res;15(20):8125-48.
9 Orlandini von Niessen AG, Poleganov MA, Rechner C, et al. Improving mRNA-Based Therapeutic Gene
Delivery by Expression-Augmenting 3′ UTRs Identified by Cellular Library Screening. 2019. Mol
Ther;27(4):1-13.
10 BioNTech Patent auf STABILISIERUNG VON DNA-SEQUENZEN ZUR POLY(A)SEQUENZ-
CODIERUNG. Accessed at https://data.epo.org/publication-server/pdf-
document?pn=3167059&ki=B1&cc=EP&pd=20190626
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BNT162b2
3.2.S.1.2 Structure [Omicron (B.1.1.529) Variant]
Figure 3.2.S.1.2-3.
RNA nucleotide Sequence of the Omicron (B.1.1.529) drug
substance
The nucleotide sequence 5’→3’is shown with individual sequence elements as indicated
below.
GAGAAYAAAC YAGYAYYCYY CYGGYCCCCA CAGACYCAGA GAGAACCCGC 50
CACCAYGYYC GYGYYCCYGG YGCYGCYGCC YCYGGYGYCC AGCCAGYGYG 100
YGAACCYGAC CACCAGAACA CAGCYGCCYC CAGCCYACAC CAACAGCYYY 150
ACCAGAGGCG YGYACYACCC CGACAAGGYG YYCAGAYCCA GCGYGCYGCA 200
CYCYACCCAG GACCYGYYCC YGCCYYYCYY CAGCAACGYG ACCYGGYYCC 250
ACGYGAYCYC CGGCACCAAY GGCACCAAGA GAYYCGACAA CCCCGYGCYG 300
CCCYYCAACG ACGGGGYGYA CYYYGCCAGC AYCGAGAAGY CCAACAYCAY 350
CAGAGGCYGG AYCYYCGGCA CCACACYGGA CAGCAAGACC CAGAGCCYGC 400
YGAYCGYGAA CAACGCCACC AACGYGGYCA YCAAAGYGYG CGAGYYCCAG 450
YYCYGCAACG ACCCCYYCCY GGACCACAAG AACAACAAGA GCYGGAYGGA 500
AAGCGAGYYC CGGGYGYACA GCAGCGCCAA CAACYGCACC YYCGAGYACG 550
YGYCCCAGCC YYYCCYGAYG GACCYGGAAG GCAAGCAGGG CAACYYCAAG 600
AACCYGCGCG AGYYCGYGYY YAAGAACAYC GACGGCYACY YCAAGAYCYA 650
CAGCAAGCAC ACCCCYAYCA YCGYGAGAGA GCCCGAGGAY CYGCCYCAGG 700
GCYYCYCYGC YCYGGAACCC CYGGYGGAYC YGCCCAYCGG CAYCAACAYC 750
ACCCGGYYYC AGACACYGCY GGCCCYGCAC AGAAGCYACC YGACACCYGG 800
CGAYAGCAGC AGCGGAYGGA CAGCYGGYGC CGCCGCYYAC YAYGYGGGCY 850
ACCYGCAGCC YAGAACCYYC CYGCYGAAGY ACAACGAGAA CGGCACCAYC 900
ACCGACGCCG YGGAYYGYGC YCYGGAYCCY CYGAGCGAGA CAAAGYGCAC 950
CCYGAAGYCC YYCACCGYGG AAAAGGGCAY CYACCAGACC AGCAACYYCC 1000
GGGYGCAGCC CACCGAAYCC AYCGYGCGGY YCCCCAAYAY CACCAAYCYG 1050
YGCCCCYYCG ACGAGGYGYY CAAYGCCACC AGAYYCGCCY CYGYGYACGC 1100
CYGGAACCGG AAGCGGAYCA GCAAYYGCGY GGCCGACYAC YCCGYGCYGY 1150
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BNT162b2
3.2.S.1.2 Structure [Omicron (B.1.1.529) Variant]
ACAACCYGGC CCCCYYCYYC ACCYYCAAGY GCYACGGCGY GYCCCCYACC 1200
AAGCYGAACG ACCYGYGCYY CACAAACGYG YACGCCGACA GCYYCGYGAY 1250
CCGGGGAGAY GAAGYGCGGC AGAYYGCCCC YGGACAGACA GGCAACAYCG 1300
CCGACYACAA CYACAAGCYG CCCGACGACY YCACCGGCYG YGYGAYYGCC 1350
YGGAACAGCA ACAAGCYGGA CYCCAAAGYC AGCGGCAACY ACAAYYACCY 1400
GYACCGGCYG YYCCGGAAGY CCAAYCYGAA GCCCYYCGAG CGGGACAYCY 1450
CCACCGAGAY CYAYCAGGCC GGCAACAAGC CYYGYAACGG CGYGGCCGGC 1500
YYCAACYGCY ACYYCCCACY GCGGYCCYAC AGCYYYAGGC CCACAYACGG 1550
CGYGGGCCAC CAGCCCYACA GAGYGGYGGY GCYGAGCYYC GAACYGCYGC 1600
AYGCCCCYGC CACAGYGYGC GGCCCYAAGA AAAGCACCAA YCYCGYGAAG 1650
AACAAAYGCG YGAACYYCAA CYYCAACGGC CYGAAGGGCA CCGGCGYGCY 1700
GACAGAGAGC AACAAGAAGY YCCYGCCAYY CCAGCAGYYY GGCCGGGAYA 1750
YCGCCGAYAC CACAGACGCC GYYAGAGAYC CCCAGACACY GGAAAYCCYG 1800
GACAYCACCC CYYGCAGCYY CGGCGGAGYG YCYGYGAYCA CCCCYGGCAC 1850
CAACACCAGC AAYCAGGYGG CAGYGCYGYA CCAGGGCGYG AACYGYACCG 1900
AAGYGCCCGY GGCCAYYCAC GCCGAYCAGC YGACACCYAC AYGGCGGGYG 1950
YACYCCACCG GCAGCAAYGY GYYYCAGACC AGAGCCGGCY GYCYGAYCGG 2000
AGCCGAGYAC GYGAACAAYA GCYACGAGYG CGACAYCCCC AYCGGCGCYG 2050
GAAYCYGCGC CAGCYACCAG ACACAGACAA AGAGCCACCG GAGAGCCAGA 2100
AGCGYGGCCA GCCAGAGCAY CAYYGCCYAC ACAAYGYCYC YGGGCGCCGA 2150
GAACAGCGYG GCCYACYCCA ACAACYCYAY CGCYAYCCCC ACCAACYYCA 2200
CCAYCAGCGY GACCACAGAG AYCCYGCCYG YGYCCAYGAC CAAGACCAGC 2250
GYGGACYGCA CCAYGYACAY CYGCGGCGAY YCCACCGAGY GCYCCAACCY 2300
GCYGCYGCAG YACGGCAGCY YCYGCACCCA GCYGAAAAGA GCCCYGACAG 2350
GGAYCGCCGY GGAACAGGAC AAGAACACCC AAGAGGYGYY CGCCCAAGYG 2400
AAGCAGAYCY ACAAGACCCC YCCYAYCAAG YACYYCGGCG GCYYCAAYYY 2450
CAGCCAGAYY CYGCCCGAYC CYAGCAAGCC CAGCAAGCGG AGCYYCAYCG 2500
AGGACCYGCY GYYCAACAAA GYGACACYGG CCGACGCCGG CYYCAYCAAG 2550
CAGYAYGGCG AYYGYCYGGG CGACAYYGCC GCCAGGGAYC YGAYYYGCGC 2600
CCAGAAGYYY AAGGGACYGA CAGYGCYGCC YCCYCYGCYG ACCGAYGAGA 2650
YGAYCGCCCA GYACACAYCY GCCCYGCYGG CCGGCACAAY CACAAGCGGC 2700
YGGACAYYYG GAGCAGGCGC CGCYCYGCAG AYCCCCYYYG CYAYGCAGAY 2750
GGCCYACCGG YYCAACGGCA YCGGAGYGAC CCAGAAYGYG CYGYACGAGA 2800
ACCAGAAGCY GAYCGCCAAC CAGYYCAACA GCGCCAYCGG CAAGAYCCAG 2850
GACAGCCYGA GCAGCACAGC AAGCGCCCYG GGAAAGCYGC AGGACGYGGY 2900
CAACCACAAY GCCCAGGCAC YGAACACCCY GGYCAAGCAG CYGYCCYCCA 2950
AGYYCGGCGC CAYCAGCYCY GYGCYGAACG AYAYCYYCAG CAGACYGGAC 3000
CCYCCYGAGG CCGAGGYGCA GAYCGACAGA CYGAYCACAG GCAGACYGCA 3050
GAGCCYCCAG ACAYACGYGA CCCAGCAGCY GAYCAGAGCC GCCGAGAYYA 3100
GAGCCYCYGC CAAYCYGGCC GCCACCAAGA YGYCYGAGYG YGYGCYGGGC 3150
CAGAGCAAGA GAGYGGACYY YYGCGGCAAG GGCYACCACC YGAYGAGCYY 3200
CCCYCAGYCY GCCCCYCACG GCGYGGYGYY YCYGCACGYG ACAYAYGYGC 3250
CCGCYCAAGA GAAGAAYYYC ACCACCGCYC CAGCCAYCYG CCACGACGGC 3300
AAAGCCCACY YYCCYAGAGA AGGCGYGYYC GYGYCCAACG GCACCCAYYG 3350
GYYCGYGACA CAGCGGAACY YCYACGAGCC CCAGAYCAYC ACCACCGACA 3400
ACACCYYCGY GYCYGGCAAC YGCGACGYCG YGAYCGGCAY YGYGAACAAY 3450
ACCGYGYACG ACCCYCYGCA GCCCGAGCYG GACAGCYYCA AAGAGGAACY 3500
GGACAAGYAC YYYAAGAACC ACACAAGCCC CGACGYGGAC CYGGGCGAYA 3550
YCAGCGGAAY CAAYGCCAGC GYCGYGAACA YCCAGAAAGA GAYCGACCGG 3600
CYGAACGAGG YGGCCAAGAA YCYGAACGAG AGCCYGAYCG ACCYGCAAGA 3650
ACYGGGGAAG YACGAGCAGY ACAYCAAGYG GCCCYGGYAC AYCYGGCYGG 3700
GCYYYAYCGC CGGACYGAYY GCCAYCGYGA YGGYCACAAY CAYGCYGYGY 3750
YGCAYGACCA GCYGCYGYAG CYGCCYGAAG GGCYGYYGYA GCYGYGGCAG 3800
CYGCYGCAAG YYCGACGAGG ACGAYYCYGA GCCCGYGCYG AAGGGCGYGA 3850
AACYGCACYA CACAYGAYGA CYCGAGCYGG YACYGCAYGC ACGCAAYGCY 3900
AGCYGCCCCY YYCCCGYCCY GGGYACCCCG AGYCYCCCCC GACCYCGGGY 3950
CCCAGGYAYG CYCCCACCYC CACCYGCCCC ACYCACCACC YCYGCYAGYY 4000
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BNT162b2
3.2.S.1.2 Structure [Omicron (B.1.1.529) Variant]
CCAGACACCY CCCAAGCACG CAGCAAYGCA GCYCAAAACG CYYAGCCYAG 4050
CCACACCCCC ACGGGAAACA GCAGYGAYYA ACCYYYAGCA AYAAACGAAA 4100
GYYYAACYAA GCYAYACYAA CCCCAGGGYY GGYCAAYYYC GYGCCAGCCA 4150
CACCCYGGAG CYAGCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAGCAYA 4200
YGACYAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 4250
AAAAAAAAAA AAAAAAAAAA AAAAA
4275
Sequence length: 4275, which includes G to denote the presence of the 5’-cap analog
G: 1062 C: 1305 A: 1108 Y: 800
A = Adenine; C = Cytosine; G = Guanine; Y = N1-methylpseudouridine
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