ESI

In-source CID

Isolate [w
5
] 2
CID

Mass Spectrum
250 500 m/z
750
Figure S1.
CID mass spectrum of m/z
-isolated [w
5
] 2 ( m/z 802) generated from in-source CID of pET Upstream DNA using a Waters Xevo TQ-S (Waters Corporation, Manchester, England,
UK). Only [phospho purine ] ions (boldface font) and precursor [w
5
] 2 ions are labeled.

ESI

Isolate [Oligo-2 – 2H] 2
CID

Mass Spectrum
[phosphoguanine] -
Ex: 230.0081
Th: 230.0085
MME: 1.7 ppm
[y
5
- G] -
Ex: 1374.2527
Th: 1374.2538
MME: 0.8 ppm
229.9
230.0
m/z
230.1
300 600
1360
900 1200 1500 m/z
1380 m/z
[y
5
- A] -
Ex: 1390.2493
Th: 1390.2488
MME: 0.4 ppm
1400
Figure S2.
CID mass spectrum of m/z
-isolated [Oligo-2 – 2H]
2 ( m/z 802; sequence: p-GTAGA) using a Linear Trap Quadrupole (LTQ) Orbitrap Discovery MS (Thermo Fisher Scientific,
Waltham, MA, USA). Instrument low mass limit of m/z 220 (for MS n experiments) prevented the inclusion of [phosphoadenine] ( m/z 214) in the CID mass spectrum; however, complementary
[y
5
– A] was observed at m/z 1390, suggesting that [phosphoadenine] was also formed but not detected. Experimental (Ex) and Theoretical (Th) masses as well as mass measurement errors
(MME, reported in ppm) for [phosphoguanine] and complementary y-B ions are labeled. Insets for m/z ranges corresponding to [phosphoguanine] ( m/z : 299.9 to 230.1) and complementary y-B ions ( m/z : 1360 to 1400) are shown at ~1000x and ~100x y-scale expansions, respectively.

Supplemental Experimental
Triple Quadrupole MS
Triple quadrupole MS experiments (Figure S1) were conducted using a Waters Xevo TQ-S
(Waters Corporation, Manchester, UK). Sample flow rate was set at 10

L/min. Cone and desolvation gas (nitrogen [N
2
]) flows were set at 150 L/min and 1000 L/min, respectively.
Nebulizer gas (N
2
) pressure was set at 7.00 bar. Desolvation and source temperatures were set at
500 °C and 150 °C, respectively. The software defined capillary, sampling cone, and source offset voltages were set at -2.5 kV, -80.0 V, and -50.0 V, respectively. Ion isolation parameters
( i.e., quadrupole rf/dc) were optimized to isolate the isotopic packet of [w
5
]
2-
( m/z 802) generated from in-source CID of pET Upstream DNA. Ions of interest were mass isolated in the first quadrupole (Q1) and fragmented in the ScanWave™ collision cell by setting a 25.0 V potential difference between Q1 and the analyzing quadrupole (Q3). Collision gas (argon) flow was set at
0.18 mL/min. The mass spectrum in Figure S1 corresponds to the average of 299 scans (5 minutes run time). Data were analyzed using MassLynx software (version 4.1) (Waters
Corporation, Manchester, UK).
Linear Trap Quadrupole (LTQ) Orbitrap MS
Orbital trap MS experiments in Figure S2 were conducted using a Linear Trap
Quadrupole (LTQ) Orbitrap Discovery MS (Thermo Fisher Scientific, Waltham, MA, USA).
Sample concentration was 1 x 10
-6
M. Doubly-charged Oligo-2 ( i.e., [Oligo-2 – 2H]
2-
) at m/z 802
was mass isolated in the LTQ and collisionally excited using a normalized collision energy [1] of
20%. Fragment ions generated in the LTQ were then sent to the Orbitrap for high mass accuracy analysis. The data in Figure S2 corresponds to the average of 50 scans. All other instrument conditions were the same as those reported for main text Figure 4.

References:
1. Lopez, L. L., Tiller, P. R., Senko, M. W., Schwartz, J. C.: Automated strategies for obtaining standardized collisionally induced dissociation spectra on a benchtop ion trap mass spectrometer. Rapid Commun. Mass Spectrom.
13 , 663-668 (1999)