Application Note

Pharma & Biopharma

Analysis of mRNA Poly-A Sequence

Variants by High-Resolution LC/MS

Author Introduction
Brian Liau
The urgency engendered by the SARS-CoV-2 pandemic of 2020 has prompted policy
Agilent Technologies, Inc.
makers and pharmaceutical firms alike to develop and deploy mRNA vaccines with
unprecedented speed. mRNA vaccines have shown impressive safety and efficacy
in clinical trials1-4, outperforming vaccines based on alternative technologies. As
mRNA vaccines are considered gene therapies5, FDA guidance requires extensive
characterization of product-related impurities. These may include populations of
mRNA molecules with slight errors in their sequence, known as sequence variants.
In addition, mRNA vaccines require lengthy, repetitive sections of A nucleotides
(poly-A) at the 3' terminus for optimal stability and biological activity.6 Both the length
and the composition of poly-A sequences are therefore critical quality attributes.
This work uses an Agilent AdvanceBio
6545XT LC/Q-TOF to analyze poly-A
tail sequences formed by E. coli Poly-A
Polymerase (PAP), which is a common
component of in vitro transcription
systems. Findings show that PAP is
not fully selective for ATP, and can act
on both CTP and UTP precursors to
incorporate significant quantities of
undesirable C and U nucleotides under
standard in vitro transcription conditions.
As these sequence variants can be
regarded as product-related impurities,
the results caution against the use of
PAP and show the value of LC/MS as
a sensitive and efficient method for
process optimization and quality control
of nucleic acid therapies.
Abbreviations used in this work:
– ATP – adenosine triphosphate
– CTP – cytidine triphosphate
– UTP – uridine triphosphate
– GTP – guanosine triphosphate
– A, C, U, and G nucleotides –
adenosine, cytidine, uridine, and
guanosine monophosphate
– Poly-A – polyadenosine
– PAP – E. coli Poly-A Polymerase
– RNA-seq – RNA sequencing
2
Experimental
In-vitro transcription of mRNA
A pCMV3 plasmid encoding a 3822 nt
gene flanked by an upstream T7
promoter and a downstream BGH
terminator sequence was purchased
from Sino Biological. The DNA sequence
was PCR amplified for 35 cycles
using T7 and BGH terminator primers
(Agilent Herculase II Fusion DNA
Polymerase, part number 600677).
After cleanup (Agilent StrataPrep PCR
Purification kit, part number 400771),
the amplified dsDNA was analyzed
on an Agilent 2100 Bioanalyzer with a
DNA 7500 kit (part number 5067-1506)
to measure its concentration and to
assess the uniformity of amplification.
The amplified dsDNA (~13 nM) was
then transcribed in vitro using a HiScribe
T7 ARCA mRNA Kit (New England
Biolabs, part number E2060S) and
tailed with the included PAP enzyme
using the manufacturer’s recommended
protocol, then precipitated with LiCl.
Aliquots of transcribed mRNA before
and after PAP tailing were analyzed on
a 2100 Bioanalyzer with an RNA 6000
Poly-A tailing experiments
A
ATP
CTP
+
UTP
RNA primer (A10)
GTP
B
ATP
CTP
+ Poly(A) +
UTP
GTP
In vitro transcribed
mRNA
Figure 1. Schematic of tailing reactions performed in this application note. (A) Reactions on RNA primer
carried out with only one precursor per reaction. (B) Reactions carried out on in vitro transcribed mRNA
under standard conditions (all precursors).
Nano kit (part number 5064-1511) to
monitor the reaction.
For PAP selectivity studies, a synthetic
10-mer poly-A sequence with 5' and
3'‑OH (Integrated DNA Technologies)
was extended with PAP enzyme
using only one precursor nucleoside
triphosphate per reaction (1 mM of either
ATP, CTP, UTP, or GTP) for 30 minutes at
37 °C, as illustrated in Figure 1A.
Sample preparation
Approximately twenty picomoles of
poly-A tailed mRNA was digested with
1,000 U of RNase T1 for 3 hours at
37 °C to liberate poly-A sequences.
Each sample was subjected to
five rounds of purification using
200 µL of oligo‑dT magnetic beads
to pull down poly-A sequences.7
Each pull‑down was eluted in 50 µL
of 1x IDTE buffer (Integrated DNA
Technologies, part number 11-05-01-05)
and pooled into a final volume of
250 µL. Prior to LC/MS analysis, the
pooled eluate was desalted into 60 µL
of deionized water using Vivaspin
500 cartridges with 10 kDa MWCO
(Sartorius, part number VS0102).
E. Coli Poly-A
Polymerase
E. Coli Poly-A
Polymerase C, U and G
LC-DAD/MS of poly-A sequences Table 1. Mobile phase and LC gradients.
Instrumentation consisted of: Agilent 1290 Infinity II LC System
– 1290 Infinity II LC with diode array Column
InfinityLab Poroshell 120 HPH-C18, 1.9 µm,
Agilent PLRP-S, 5 µm, 2.1 × 50 mm, 1,000 Å
2.1 × 50 mm,120 Å
detector (P/N G7117B)
Solvent A 15 mM dibutylamine + 25 mM HFIP in DI water
– 6545XT AdvanceBio LC/Q-TOF Solvent B 15 mM dibutylamine + 25 mM HFIP in methanol
Care was taken to eliminate glass from 0 to 2 min, 15% B 0 to 1 min, 15% B
Gradient 12 min, 30% B 10.5 min, 45% B
the flow path to reduce alkaline metal 12.1 to 13 min, 90% B 10.6 to 11.5 min, 90% B
adduction. Agilent Nalgene bottles Column Temperature 50 °C 80 °C
(part number 9301-6460) were used
Flow Rate 0.4 mL/min
as mobile phase containers, and each
Injection Volume 10 to 20 µL
solvent line was equipped with a steel
frit. Agilent polypropylene sample vials Table 2. Mass spectrometer settings.
were used (part number 5190-2242).
Agilent 6545XT AdvanceBio LC/Q-TOF
Before first use, the LC system and
LC/MS LC/MS/MS
column were flushed with a 50% MeOH
Acquisition Mode Negative, standard (3,200 m/z) mass range, high sensitivity (2 Ghz)
+ 0.1% formic acid solution overnight to
Gas Temperature 350 °C
further reduce alkaline metal adducts.
Gas Flow 12 L/min
If required, a 30-minute flush with
Nebulizer 55 psig
50% MeOH + 0.1% formic acid was
Sheath Gas Temperature 275 °C
usually enough to clean the system
between experiments.8 Sheath Gas Flow 10 L/min

Vcap 4,500 V
Poly-A sequences were separated on
Nozzle Voltage 2,000 V
a PLRP-S column (2.1 × 50 mm, 5 µm,
Fragmentor 250 V
1,000 Å, part number PL1912-1502).
Skimmer 65 V
To achieve higher chromatographic
MS1 Range 400 to 3,200 m/z
resolution, an Infinity Poroshell 120
MS1 Scan Rate 2 Hz 5 Hz
HPH-C18 column (2.1 × 50 mm, 1.9µm,
MS2 Range 50 to 3,200 m/z
120 Å, part number  699675-702) was
MS2 Scan Rate 3 Hz
used in PAP selectivity experiments.
MS2 Isolation Width Medium (~4 amu)
The mobile phase and LC gradients
are shown in Table 1. The mass Collision Energy 0, 40, 60 V
On; 3 repeat then exclude for
spectrometer was operated in negative Threshold for MS2
0.2 min
ion mode with settings in Table 2,
Precursor Abundance
and data analysis was performed in Based Scan Speed
N/A Yes

BioConfirm 10.0 with deconvolution Target (Counts/Spectrum) 25,000

settings in Table 3. Use MS2 Accumulation
Yes
Time Limit
Purity 100% stringency, 30% cutoff

Sort Precursors By abundance only; +3, +2, +1

Reference Mass 1,033.9881

Table 3. Deconvolution settings.

Agilent MassHunter BioConfirm B10.0 Settings

Oligonucleotide Length ≤30 nt ≥90 nt

Deconvolution Algorithm Maximum Entropy

Subtract Baseline 1

Adduct Proton loss

Mass Range 3,000 to 10,000 Da 30,000 to 60,000 Da

Mass Step 0.05 Da 0.05 Da

Use Limited m/z Range 1,040 to 3,200 800 to 2,500

3
 Results and discussion
This first test analyzed poly-A sequences
extended by PAP on a synthetic RNA
primer consisting of 10 repeated
A nucleotides (A10) in the presence of
1 mM ATP. As shown in Figure 2 this
resulted in a bimodal distribution of
poly-A sequences, with one population
consisting of shorter oligonucleotides
eluting from 2.5 to 6 minutes, and
another consisting of longer nucleotides
eluting in a broad peak at ~10.6 minutes.
Mass spectrometric analysis indicated
the shorter population ranged in size
from 11 to 22 nt (Figure 3D), whereas
the longer population ranged from
108 to 149 nt in length (35,492.89 to
48,990.17 Da, Figure 4C).
Extracted and deconvoluted mass
spectra from three selected peaks
from the shorter oligonucleotide
population are shown in Figure 3. Mass
spectra consisted primarily of doubly
and triply charged ions generated
through proton loss, as well as minor
populations of sodium adducts.
Isotopically resolved deconvoluted mass
spectra were assigned identities of
A20, A21, and A22 (Figures 3B to 3D) with
<5 ppm error based on the respective
monoisotopic peaks.

×102
5.0 A RNA Primer + ATP A108 to A149
4.5 UV: λ = 260 nm
4.0 10.675

3.5
Response

3.0
2.5 A11 to A22
2.0
1.5
3.184
1.0 2.879
3.489
2.555 3.812
0.5 4.207 4.599
4.957 5.275 5.830
0

×108
B 10.639
1.0
Total ion current
0.9
0.8
Response

0.7
0.6
0.5
0.4
0.3
0.2
2.549 2.865 3.172 3.472 3.796
0.1 4.187 4.578 4.952 5.268
5.817
0
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0
Acquisition time (min)

Figure 2. UV absorbance at 260 nm (A: reference = 360 nm) and total ion chromatogram (B) of RNA primers extended with PAP in the presence of only ATP.
Separation was carried out on a PLRP-S column.

4
 ×101 ×104 ×104
A TIC: A11 to A22 1.4 B Peak 1: A20 6521.13 Deconvoluted
1.5 1.8
1.4 1.3 1.6

2172.69121
1.2 1.4 6523.15
1.3 2.865 3.172 [M–3H]3–
3.472 6520.13
1.1 1.2

Counts
1.2

2173.36040
2.549 1.0 1.0
1.1 6524.15

2172.35710
0.8
Response (%)

0.9
1.0 3.796 0.6

Counts
6519.12 6525.15
0.8
0.9 0.4

2173.69437
6526.16
0.7 6527.16
0.8 4.187 0.2 6529.15
0.6
0.7 0

2172.02329
4.578 0.5 6518 6520 6522 6524 6526 6528
0.6 Peaks
0.4 Deconvoluted mass (amu)
0.5 4.952 1
2 3 0.3
0.4 5.268 [M–4H+Na]3–
0.2
0.3 5.550 [M–5H+2Na]3–
5.817 *6.066 0.1
0.2
0
2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 2170 2172 2174 2176 2178 2180 2182 2184 2186 2188 2190
Acquisition time (min) Mass-to-charge (m/z)

×103 ×103 ×103 ×103

C Peak 2: A21 6851.18 Deconvoluted D Peak 3: A22 7 Deconvoluted
7.5 9 5.2 7180.23
7.0 8 6852.19 6 7179.22
2282.70967

4.8

2392.39270
6.5 7181.25
7 [M–3H]3– 5
6849.18 4.4
6.0 6
Counts

Counts
[M–3H] 3– 7178.23

2393.06199
6853.20 4.0 4
5.5 5 7182.24
5.0 4 3.6 3
2282.04118

6854.21 7183.25

2391.72447
Counts

Counts

4.5 3 6848.16 3.2

2283.37684

2 7184.26
4.0 2 6855.21 7177.22
2.8 7185.25
1
6856.21 1
3.5 6858.24 2.4 7186.31
3.0 0 0
6848 6850 6852 6854 6856 6858 2.0 7178 7180 7182 7184 7186
2.5
Deconvoluted mass (amu) 1.6 Deconvoluted mass (amu)
2.0
1.2
1.5
[M–4H+Na]3– 0.8 [M–4H+Na]3–
1.0
[M–5H+2Na]3– [M–5H+2Na]3–
0.5 0.4
0 0
2280 2282 2284 2286 2288 2290 2292 2294 2296 2298 2300 2390 2392 2394 2396 2398 2400 2402 2404 2406 2408 2410
Mass-to-charge (m/z) Mass-to-charge (m/z)

Figure 3. A11 to A 22 oligonucleotides formed by PAP. (A) Total ion current chromatogram showing the three selected peaks with extracted mass spectra shown
in (B to D). Deconvoluted mass spectra of A 20 (Mobs = 6,519.12 Da, Mtheo = 6,519.09 Da), A 21 (Mobs = 6,848.16 Da, Mtheo = 6,848.15 Da), and A 22 (Mobs = 7,177.22 Da,
Mtheo = 7,177.20 Da) are shown as insets. Mobs: observed monoisotopic mass; Mtheo: theoretical monoisotopic mass.

A portion of the longer oligonucleotides A nucleotides, increasing the theoretical additions of up to two monomers
was sampled for deconvolution average mass by 329.209 Da. Table 4 of C nucleotides (Figure 5A) or one
(Figure 4A). The charge envelope in shows that mass peaks in Figure 4D U nucleotide (Figure 5B) to the RNA
the extracted mass spectra from 10 were confidently annotated as A121 to A138 primer, indicating that PAP was not
to 10.3 minutes primarily fell between with differences between theoretical and wholly selective for ATP. The addition
800 to 2,500 m/z (Figure 4B) and was observed masses ≤1.16 Da. of guanosine monophosphate was not
deconvoluted to a destination mass To assess the selectivity of PAP for ATP, observed in this experiment (Figure 5C)
range of 30 to 60 kDa. The deconvoluted duplicate experiments were conducted but could not rule out the possibility that
mass spectra (Figure 4C) clearly where PAP was added to the RNA appreciable quantities might be added
showed a heterogenous population of primer in the presence of only 1mM CTP, with longer reaction times or higher GTP
sample peaks from 34 to 50 kDa, which UTP, or GTP. Although the extension concentrations. Overall, PAP showed the
were evenly separated by 329.2 ±1 Da of long polymeric chains were not highest activity with ATP, followed by
(Figure 4D). These mass increments observed, chromatographically resolved CTP, UTP, and GTP in descending order.
were consistent with single additions of

5
 ×102 ×103 524.96535 1033.98812
A TIC: A108 to A149 2.8
1.0
10.639 B 966.00006 Extracted MS: 10 to 10.3 minutes
2.6
0.9 2.4
723.90809
2.2
0.8
2.0
Response (%)

0.7 1.8

Counts
0.6 Deconvolute 1.6
10 to 10.3 minutes 1.4
0.5
1.2
0.4
1.0
0.3 0.8
0.2 0.6
0.4
0.1
0.2
0 0
9.3 9.5 9.7 9.9 10.1 10.3 10.5 10.7 10.9 11.1 400 600 800 1000 1200 1400 1600 18002000 2200 2400 2600 2800 3000
Acquisition time (min) Mass-to-charge (m/z)

×102 ×102
3.4 C Deconvoluted MS 3.6 D Deconvoluted MS (zoomed)
3.2 3.4
3.0 41418.82 3.2 329.36 329.56
2.8 3.0 329.71 41418.82
328.49 329.56
2.6 2.8 328.66

2.4 39114.50 45040.68 2.6 328.74

329.30
2.2 2.4 43393.78
329.85
2.0 2.2 328.68 329.28 329.52
Counts
Counts

40101.53
1.8 2.0 39773.03 42406.35
329.17 328.61
42735.87 328.73
1.6 1.8 41748.39
40759.75 43723.09 44381.54
1.4 51398.77 55306.54
1.6 43065.04
44710.28
1.4 40431.09 44052.93
1.2
1.2
49974.73

1.0
57539.28 1.0
0.8 52717.55 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 0
32000 36000 40000 44000 48000 52000 56000 40000 40500 41000 41500 42000 42500 43000 43500 44000 44500
Deconvoluted mass (amu) Deconvoluted mass (amu)

Figure 4. A108 to A149 oligonucleotides formed by PAP. (A) Total ion current chromatogram showing the region 10 to 10.3 minutes sampled for deconvolution.
(B) Charge envelope and (C) Deconvoluted mass spectrum of sampled region. Dashed arrows (left = 35,492.89 Da, right = 48,990.17 Da) indicate the range of
mass peaks that could be confidently assigned identities A108 to A149. (D) Enlarged deconvoluted mass spectrum showing regular intervals between peaks from
39,773.03 to 44,710.28 Da.

Table 4. Annotated mass peaks from Figure 4D.

Oligonucleotide Observed Mass (Da) Theoretical Mass (Da) Mass Difference (Da)
A121 39,773.03 39,772.08 0.95

A122 40,101.53 40,101.28 0.25

A123 40,431.09 40,430.49 0.6

A124 40,759.75 40,759.70 0.05

A125 41,089.46 41,088.90 0.56

A126 41,418.82 41,418.11 0.71

A127 41,748.39 41,747.32 1.07

A128 42,077.06 42,076.52 0.54

A129 42,406.35 42,405.73 0.62

A130 42,735.87 42,734.94 0.93

A131 43,065.04 43,064.15 0.89

A132 43,393.78 43,393.35 0.43

A133 43,723.09 43,722.56 0.53

A134 44,052.93 44,051.77 1.16

A135 44,381.54 44,380.97 0.57

A136 44,710.28 44,710.18 0.1

6
 RNA Primer + CTP, UTP, or GTP
×103
A A10
1.0 +GTP 4.158

0.8
Rresponse

0.6

0.4

0.2

×103
B A10
1.0
+UTP 4.155
0.8
Rresponse

0.6

0.4

0.2 A10U
4.285
0

×102
9 C A10
8 +CTP 4.117
7
6
Rresponse

5
4
3
A10C
2 4.387
A10CC
1
5.052
0
4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4

Acquisition time (min)

D

Figure 5. UV absorbance (Abs = 260 nm, Ref = 360 nm) chromatograms showing promiscuous activity of PAP towards (A) GTP, (B) UTP, and
(C) CTP. No addition of guanosine monophosphate was detected. (D) Relative quantitation of A10, A10C, and A10CC as shown in panel (C). Separation
was carried out on an Agilent Poroshell 120 HPH-C18 column.

7
 The deconvoluted mass spectra of and U nucleotides were indeed added Next, full-length, in vitro transcribed
the unmodified RNA primer and those to the 3' terminus of the RNA primer mRNA were analyzed on a Bioanalyzer
extended with C or U nucleotides are (Figure 7), resulting in the formation of equipped with RNA 6000 Nano kit and
shown in Figure 6. Isotopically resolved characteristic doubly charged y-ions by LC/MS. Before tailing, transcribed
deconvoluted mass spectra were 1601.271 m/z and 1601.758 m/z. In mRNA showed the expected length of
assigned identities of A10, A10C, A10CC contrast, the unmodified RNA primer was ~3,800 nt which increased to ~4,200 nt
and A10U with <13 ppm error based on terminated with a 3' A nucleotide, yielding after reaction with PAP (Figure 8A),
the respective monoisotopic peaks. a doubly charged y-ion 1448.749 m/z indicating that successful poly-A tailing
MS/MS experiments showed that C upon fragmentation. had been achieved.

×106 ×106
×106 2.4 Deconvoluted ×105
A A10 1.4 Deconvoluted
8.0 B
1.1 3229.61 A10C 3534.66
2.2
2.0 7.5 1.2 3533.65
1.0
1613.77898

1766.29964
1.8 3228.61 3230.61
1.6 7.0 1.0
0.9 [M–2H]2– 1.4 6.5 0.8 3536.66
1.2 3231.61 [M–2H]2–
0.8 1.0 6.0 0.6
1614.28019
1613.27764

0.8 5.5
Response (%)

1765.79879
0.4 3537.66
0.7 0.6 3232.61
5.0
Counts
0.4 3538.66
3233.61 0.2 3539.65
0.6 0.2 3235.61 4.5

1767.30299
0 4.0 0
3228 3230 3232 3234 3236 3533 3535 3537 3539 3541
1614.78179

0.5 3.5
Deconvoluted mass (amu) [M–3H+Na]2– Deconvoluted mass (amu)
0.4 [M–3H+Na]2– 3.0

1767.80368
2.5

1777.28922
0.3
1624.76866

2.0

1778.29111
1615.78384

0.2 1.5
1.0
0.1 [M–4H+2Na]2– [M–4H+2Na]2–
0.5
0 0
1612 1616 1620 1624 1628 1632 1636 1762 1766 1770 1774 1778 1782 1786 1790 1794
Mass-to-charge (m/z) Mass-to-charge (m/z)

×104 ×105 D A10U ×105

C A10CC 3839.68 ×105
1.6 3535.63
6.5 Deconvoluted 1.5 2.4 Deconvoluted
1.4 3840.68 3536.63
1766.79116

6.0
1918.81951

1.2 1.4 2.0 3534.62

3838.67 1.3
5.5 1.0 1.6
3841.68 3537.63
5.0 0.8 1.2
[M–2H]2– 1.2
0.6 1.1
1766.28933

4.5 3842.68 [M–2H] 2–

0.8
0.4 1.0 3538.63
1918.31778

4.0 3843.68 0.4 3539.63

0.9
1919.82144
Counts

Counts

0.2 3844.68 3540.62

3.5
1767.79274

0 0.8 0
3.0 3838 3840 3842 3844 3846 0.7 3534 3536 3538 3540 3542
Deconvoluted mass (amu)
1778.29427

Deconvoluted mass (amu) 0.6

1920.32232

2.5
1768.29394
1929.80855

[M–4H+2Na]2– 0.5 [M–4H+2Na]2–

2.0
1777.27869
1930.81204

1779.29906

0.4
1788.77708
1940.79893

1.5
0.3
1.0 [M–3H+Na]2–
[M–3H+Na]2– 0.2
0.5 0.1
0 0
1918 1920 1922 1924 1926 1928 1930 1932 1934 1936 1938 1940 1942 176417661768177017721774177617781780178217841786178817901792
Mass-to-charge (m/z) Mass-to-charge (m/z)

Figure 6. Extracted and deconvoluted mass spectra of (A) Unmodified A10 RNA primer (Mobs = 3,228.61 Da, Mtheo = 3,228.57 Da), (B) Extended with one C nucleotide
(Mobs = 3,533.65 Da, Mtheo = 3,533.61 Da), (C) Extended with two C nucleotides (Mobs = 3,838.67 Da, Mtheo = 3,838.65 Da), (D) Extended with one U nucleotide
(Mobs = 3,534.62 Da, Mtheo = 3,534.59 Da). Mobs: observed monoisotopic mass; Mtheo: theoretical monoisotopic mass.

8
 Counts Counts Counts

×10

0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0
0.2
0.4
0.6
0.8
1.0
1.2
0
0.5
1.0
1.5
2.0
2.5

×103
×103
3
81.66265 96.97037 75.69614

10
A10
159.53278
188.55510

A10U
1.4 A C
265.79711 241.61366
329.05013 351.57370 346.05725
408.01125
438.19732
485.06727 504.03294 522.04290
572.11096 571.13036 595.14191
657.09966 657.09908 657.09773
692.76108
771.13111 777.01508 771.12407
851.09472
901.16328 900.18568 924.19419
986.14701 986.14634 986.14926
1069.43200
1100.18019 1100.18108
MS/MS of RNA oligonucleotides

1168.62726
1230.21069 1229.23464 1253.24944
1315.20047 1315.20260 1315.20392
1383.46045 1381.72562
1448.75397
1504.14858 1480.22444
1546.25261
1602.76457 1601.77530
1699.25717 1698.77512 1613.78124
1758.27000
1766.78907 1766.30071 1838.25219
1889.32922 1888.33382 1912.34499
1973.30595 1975.06167 1974.31090

Mass-to-charge (m/z)
Mass-to-charge (m/z)
Mass-to-charge (m/z)

2092.23546 2074.62158 2088.32333

2167.29222
2217.35839 2217.39990 2240.40774
2304.62440 2303.36525
2342.41332 2368.51287
2434.74789 2439.88899 2460.91200
2548.43473 2541.46701 2569.46422
2632.45564 2657.23576 2632.43453
2727.18466 2702.68802
2772.83640
2845.47504 2817.98975
2864.15346
2911.44590
2961.43148 2961.48408
3036.67675 3042.03588 3032.88026
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
3101.57393
3152.75440 3143.12122

0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0

×102
×102

Figure 7. MS/MS of selected oligonucleotides showing diagnostic ions characteristic of their different 3' termini.
1447.00817
1447

1600.68613 1447.24666
1600.5

1600.99275 1447.71859
1601.27066
1448

1601.56487 1448.24668
1601.75870 1601.77323
1601.5

1601.89486 1448.74940
1602.06985 1448.91784
1602.27340 1602.16836 1449.12714
1602.26999 1449.25277
1449.39321
1602.51450
1602.65440 1449.75383
1602.76457 1602.69462 1602.77801
1449.93645
1603.01776
1603.12558 1450.25583
1603.27041 1603.26362 1450.42005
1603.56812 1450.74813
1603.74940 1603.78754 1450.97516
1603.92994 1603.94883 1451.25462
1604.16436 1451.44059
Mass-to-charge (m/z)
Mass-to-charge (m/z)
Mass-to-charge (m/z)

×102 3’ diagnostic y-ion 1,601.758 (z = –2)

3’ diagnostic y-ion 1,601.271 (z = –2)
3’ diagnostic y-ion 1,448.749 (z = –2)

1604.26304 1604.35725 1451.73856

1604.44230 1604.57208 1452.05549
1449 1450 1451 1452

1604.77938
1602.5 1603.5 1604.5

1452.39800
1600.5 1601.5 1602.5 1603.5 1604.5

1604.94142 1605.05783 1452.64745

1605.24651 1453.03120
1605.56409

9
1453.22916
1453

1605.50056
1605.5
Full length mRNA samples were digested
with RNase T1, followed by repeated
pull-downs with oligo dT magnetic
beads to yield purified tail sequences.
As with PAP-extended RNA primers,
tail sequences derived from in vitro
transcribed mRNA consisted of both a
shorter population of oligonucleotides
eluting between 3.7 to 7.5 minutes and a
longer population eluting ~10.6 minutes
(Figure 8B). Extracted and deconvoluted
mass spectra from selected peaks in
the shorter population revealed poly-A
sequences ranging in length from 16 to
27 nt, with each containing a single
misincorporated U nucleotide (Figure 9).
Although not seen in this dataset,
misincorporated C nucleotides were also
observed in other experiments.
As noted by M. Beverly et al.7, tail
sequences formed by PAP are
considerably more heterogenous in
length as compared to genetically
templated poly-A sequences. The
results indicate that this heterogeneity is
compounded by the misincorporation of
differing numbers of C and U nucleotides
when the tailing reaction takes place
under standard conditions with all four
precursor nucleoside triphosphates
present, making the mass spectra of
longer tail sequences very challenging
to deconvolute.

Bioanalyzer RNA 6000 Nano Kit ×102

1.1 B 10.644
A 1.0
In vitro transcribed mRNA
T7 + PAP

0.9
T7 Only
Ladder

0.8 UV: λ = 260 nm

Response

0.7
0.6
0.5 (A16 to A27) + U
0.4
0.3
0.2 4.444
3.030 3.760 5.129 5.680 7.260 7.779
0.1 2.785 3.420 4.120 4.797 5.407 5.947 6.379 6.932 7.535 8.152 8.710
0
×102
10.625
1.0
Total ion current
0.9
6000 0.8
Response

4000 0.7
0.6
2000 0.5
1000 0.4
500 0.3
200 0.2 4.089 4.780 5.370 6.384
25 0.1 3.740 4.430 5.129 * 5.919 8.072
0
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0
Acquisition time (min)

Figure 8. (A) Bioanalyzer analysis of in vitro transcribed mRNA before (lane 1) and after (lane 2) tailing with PAP. (B) UV absorbance at 260 nm (top panel) and
total ion chromatogram (bottom panel) of poly-A sequences appended to in vitro transcribed mRNA in the presence of all four nucleoside phosphate precursors.
Separation was carried out on a PLRP-S column.

10
 ×103 ×104
5.0 A TIC: (A16 to A27) + U B Peak 1: A18 + U Deconvoluted
5.5 1.0 6169.05

6171.05
4.5 0.9
5.0 0.8

2055.33118
4.0 0.7 6168.04

6172.05
4.5 [M–3H]3–

2055.66660
0.6

6173.05
3.5

6175.05
4.0 0.5

2054.99683

6177.05
0.4 6167.04

2055.99955
3.0
3.740 3.5 0.3
Response

2.5 Peaks 0.2

Counts
3.0 0.1
2.0 4.089 1 2 3 *5.919 0
4.430 4.780 5.129 5.370 *5.678 *6.168 6.384 2.5 6166 6170 6174 6178
1.5
2.0 Deconvoluted mass (amu)
1.0 [M–4H+Na]3–
1.5
0.5
1.0
0
0.5
-0.5
0
3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 2054 2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065 2066
Acquisition time (min) Mass-to-charge (m/z)

×103 ×103 ×103 ×103

C Peak 2: A19 + U Deconvoluted D Peak 3: A20 + U Deconvoluted
5.0 8 8
6499.11 5.5 6828.16
7 7
4.5
2165.34897

5.0 [M–3H]3– 6 6829.16

2165.01600

6
4.0 [M–3H]3– 6497.10 6500.11
5 4.5 5 6826.16

2275.03350
6830.17

2274.69989
4 4
2165.68441

3.5 6501.11 4.0

2164.68107

2275.36796
3 6502.12 3 6831.16
3.0 6496.10 3.5 6833.16
Counts

Counts

2 6503.10 2 6825.15
2.5 1 3.0 1
0 2.5 0
2.0 6496 6498 6500 6502 6504 6824 6826 6828 6830 6832 6834
Deconvoluted mass (amu) 2.0 Deconvoluted mass (amu)
1.5
1.5 [M–4H+Na]3–
1.0 [M–4H+Na]3–
1.0
0.5 0.5
0 0
2164 2165 2166 2167 2168 2169 2170 2171 2172 2173 2174 2175 2176 2274 2275 2276 2277 2278 2279 2280 2281 2282 2283 2284 2285
Mass-to-charge (m/z) Mass-to-charge (m/z)

Figure 9. A16 to A 27 oligonucleotides with misincorporated U nucleotide. (A) Total ion current chromatogram showing three selected peaks with extracted mass
spectra shown in (B to D). Deconvoluted mass spectra of A18 + U (Mobs = 6,167.04 Da, Mtheo = 6,167.01 Da), A19 + U (Mobs = 6,496.10 Da, Mtheo = 6,496.07 Da), and
A 20 + U (Mobs = 6,825.15 Da, Mtheo = 6,825.12 Da) are shown as insets. Mobs: observed monoisotopic mass; Mtheo: theoretical monoisotopic mass.

Conclusion Although these sequence variants To achieve such sensitivity and

may be inconsequential for in vitro selectivity, one prior study demonstrated
This study shows that: (1) the intact studies, they are highly significant from PAP’s off-target activity by using
masses of long (121 to 136 nt), a regulatory standpoint. Notably, other radiolabeled nucleotides.10 Such
heterogenous poly-A sequences can be in vitro transcription enzymes such techniques can be hazardous and are
accurately measured by deconvolution as T7 polymerase may also produce ill-suited for production environments.
of their ensemble mass spectra, and (2) sequence variants through mechanisms LC/MS can achieve single-nucleotide
PAP is not fully selective for ATP under such as slippage or transcriptional selectivity without the need for such
standard in vitro transcription conditions, arrest9, underscoring the need for highly reagents. Moreover, LC/MS can detect
causing both C and U nucleotides to be sensitive and selective methods for and quantify sequence variants without
added to poly-A tail sequences. detecting these impurities. the lengthy reverse transcription, ligation
and amplification steps characteristic of
RNA-seq, which are known to introduce
biases and artifacts.

11
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5994-3005EN
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