Structural elucidation of bisulfite adducts to pseudouridine that result in deletion signatures during reverse transcription of RNA

Article abstract of DOI:10.1021/jacs.9b08630

The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications N¹-methyladenosine, 5-methylcytosine, and pseudouridine (ψ) via bisulfite treatment of RNA provides a key advance to locate these important modifications. The locations of ψ were found by a deletion signature generated during cDNA synthesis after bisulfite treatment for which the chemical details of the reaction are poorly understood. In the present work, the bisulfite reaction with ψ was explored to identify six isomers of bisulfite adducted to ψ. We found four of these adducts involved the heterocyclic ring, similar to the reaction with other pyrimidines. The remaining two adducts were bonded to the 1′ carbon, which resulted in opening of the ribose ring. The utilization of complementary 1D- and 2D-NMR, Raman, and electronic circular dichroism spectroscopies led to the assignment of the two ribose adducts being the constitutional isomers of an S- and an O-adduct of bisulfite to the ribose, and these are the final products after heating. A mechanistic proposal is provided to rationalize chemically the formation and stereochemistries of all six isomeric bisulfite adducts to ψ conversion of intermediate adducts to the two final products is proposed to involve E2, S_N2′, and [2,3]-sigmatropic shift reactions. Lastly, a synthetic RNA template with ψ at a known location was treated with bisulfite, leading to a deletion signature after reverse transcription, supporting the RBS-Seq report. This classical bisulfite reaction used for epigenomic and epitranscriptomic sequencing diverges from the C nucleoside ψ to form stable bisulfite end products that yield signatures for next-generation sequencing.

Full text of DOI:10.1021/jacs.9b08630

Subscriber access provided by Eastern Michigan University | Bruce T. Halle Library
Article
Structural elucidation of bisulfite adducts to pseudouridine that
result in deletion signatures during reverse transcription of RNA
Aaron M. Fleming, Anton Alenko, Jay P. Kitt, Anita M Orendt,
Peter F. Flynn, Joel M. Harris, and Cynthia J. Burrows
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08630 • Publication Date (Web): 20 Sep 2019
Downloaded from pubs.acs.org on September 21, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
Structural elucidation of bisulfite adducts to pseudouridine that result
in deletion signatures during reverse transcription of RNA
7
8
9
Aaron M. Fleming,† Anton Alenko,† Jay P. Kitt,† Anita M. Orendt,†‡ Peter F. Flynn,†
Joel M. Harris,† and Cynthia J. Burrows†*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†Department of Chemistry, University of Utah, Salt Lake City, UT 84112-0850, United States
‡Center for High Performance Computing, University of Utah, Salt Lake City, UT 84112-0190,
United States
*To whom correspondence should be addressed burrows@chem.utah.edu
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 31
1
2
3
4
Abstract
5
6
7
8
9
The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications
N1-methyladenosine, 5-methylcytosine, and pseudouridine (Ψ) via bisulfite treatment of RNA
provides a key advance to locate these important modifications. The locations of Ψ were found
by a deletion signature generated during cDNA synthesis after bisulfite treatment for which the
chemical details of the reaction are poorly understood. In the present work, the bisulfite reaction
with Ψ was explored to identify six isomers of bisulfite adducted to Ψ. We found four of these
adducts involved the heterocyclic ring, similar to the reaction with other pyrimidines. The
remaining two adducts were bonded to the 1‵ carbon, which resulted in opening of the ribose
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ring. The utilization of complementary 1D- and 2D-NMR, Raman and electronic circular dichroism
spectroscopies led to the assignment of the two ribose adducts being the constitutional isomers
of an S- and an O-adduct of bisulfite to the ribose, and these are the final products after heating.
A mechanistic proposal is provided to rationalize chemically the formation and stereochemistries
of all six isomeric bisulfite adducts to Ψ; conversion of intermediate adducts to the two final
products is proposed to involve E2, SN2’ and [2,3]-sigmatropic shift reactions. Lastly, a synthetic
RNA template with Ψ at a known location was treated with bisulfite leading to a deletion signature
after reverse transcription, supporting the RBS-Seq report. This classical bisulfite reaction used
for epigenomic and epitranscriptomic sequencing diverges from the C nucleoside Ψ to form stable
bisulfite end products that yield signatures for next-generation sequencing.
2
ACS Paragon Plus Environment

Page 3 of 31
Journal of the American Chemical Society
1
2
3
4
Introduction
5
6
7
8
9
Bisulfite addition to carbonyl-containing compounds is a classical reaction in organic
chemistry that has revolutionized our understanding of biology. Historically, reversible bisulfite
addition to aldehydes or ketones was a convenient method for purification of organic compounds
by recrystallization of the bisulfite adduct.1 While modern chromatographic techniques have
displaced this reaction from organic chemistry textbooks, bisulfite chemistry finds application
today in its conjugate addition reaction to carbon-6 of cytidine. In 1970, Shapiro and Hayatsu
independently observed that under acidic conditions bisulfite adds to the heterocyclic ring in 2‵-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
deoxycytidine (dC) resulting in selective deamination of the exocyclic amine to furnish 2‵-
deoxyuridine (dU) upon desulfonation (Scheme 1A).2,3 In 1980, Ehrlich demonstrated that 5-
methyl-2‵-deoxycytidine (5mC) reacts ~50-fold slower with bisulfite than dC.4 Finally, in 1992
Frommer, et al. applied the bisulfite reaction to DNA to map the locations of the epigenetic mark
5mC.5 Bisulfite treatment of DNA enabled sequencing for 5mC because the dC bases would
preferentially react and deaminate to be sequenced as thymidine (dU equivalent) in the PCR
amplicons, while the 5mCs continued to code like dC. The relative ease in conducting the bisulfite
reaction has resulted in this method being applied to high-throughput sequencing.6,7 Hybrid
approaches using the bisulfite reaction were developed to sequence the TET-mediated oxidation
products of 5mC that include 5-hydroxymethyl-2`-deoxycytidine,8,9 5-formyl-2`-deoxycytidine,10,11
and 5-carboxyl-2`-deoxycytidine.12 Many additional chemoselective tags have been developed
for sequencing the 5mC oxidation products in DNA.13,14 Thus, bisulfite chemistry opened the door
for many discoveries to be made regarding how epigenetic modification of dC in particular regions
of the genome impacts cellular phenotype.15
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 31
1
2
3
4
5
A
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B
Scheme 1. (A) The bisulfite reaction with dC/C yields dU/U that codes like T. (B) The bisulfite
adduct to Ψ as reported by Singhal16 or Shapiro and Everett.17
Decoration of RNA with site-specific modifications is well established.18-20 More recent
findings have identified that RNA modifications, particularly in mRNA, can alter the coding
potential, cellular half-life, alternative splicing, and compartmentalization of these coding strands,
opening the field of epitranscriptomics.21-23 Chemical modifications to RNA alter its physical
properties and impact secondary structure or change the coding potential of the bases.24-26
Prominent regulatory modifications in mRNA include N6-methyladenosine (m6A), N1-
methyladenosine (m1A), 5-methylcytidine (m5C), inosine (I), and pseudouridine (Ψ); all of these
appear to function together at various stages in regulating RNA biogenesis.18,21-23,27 Enabling
chemical tools for sequencing RNA modifications include glyoxal and RNase T1 to generate site-
specific strand cleavage at I that is identified during sequencing;28 metabolic incorporation of
photoreactive 5-thiouridine to trap m6A-specific antibodies for RNA enrichment and sequencing;29
bisulfite chemistry to sequence m5C in RNA or other chemoselective tags;14,30 and N-cyclohexyl-
N‵-(2-morpholinoethyl)-carbodiimide (CMC) or its derivatives react specifically with Ψ to generate
4
ACS Paragon Plus Environment

Page 5 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
a stop in cDNA synthesis observed during sequencing.31-35 Recently, we assisted the Cairns
laboratory to discover RBS-Seq, a bisulfite-based reaction on RNA that permits sequencing of
three RNA modifications simultaneously, including m1A, m5C, and Ψ.36 Beyond the expected
m5C result, m1A was located via Dimroth rearrangement to m6A during bisulfite treatment that
converted a cDNA synthesis stop to a bypass, and Ψ was found by a deletion signature following
reverse transcription. The ongoing mystery with this method was why does bisulfite treatment of
Ψ result in a deletion signature during cDNA synthesis?
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In 1974, Singhal studied the bisulfite reaction with the Ψ nucleoside and identified two
stable product peaks that retained the aromatic UV-vis absorbance at ~265 nm, similarly to Ψ,
and the products formed a vicinal-diol borate complex supporting the conclusion that the ribose
sugar was still present.16 Curiously, Singhal concluded bisulfite formed a stable adduct to the
heterocycle (Scheme 1B), which would no longer be aromatic, a conclusion inconsistent with the
UV-vis data. The only other report of this reaction comes from a Ph.D. dissertation from Shapiro’s
laboratory (Donald W. Everett, Ph.D.; NYU, 1980).17 The bisulfite reaction on the Ψ nucleoside
was found to yield two products that had the same UV-vis spectra as reported by Singhal. Further,
1
the products had masses consistent with mono-bisulfite adducts, while the H-NMR for the two
products were different with respect to the 1‵ proton signals, and the optical rotary dispersion
spectra were mirror images of one another with subtle peak shape differences. Everett and
Shapiro proposed that bisulfite was adducted to a ribose ring-opened nucleoside at the 1‵ carbon
yielding a new carbon to sulfur covalent bond, and the two peaks represented diastereomeric
products (Scheme 1B); however, the results were never published.
In the present report, we harnessed modern spectroscopic methods to discover that
bisulfite reacts with the Ψ nucleoside to form two stable aromatic end products, both of which are
mono-bisulfite adducts at C1’ and have a ring-opened ribose. However, instead of being a pair of
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 31
1
2
3
4
5
6
7
8
9
diastereomers, these two products are constitutional isomers in which one is an O-adduct while
the other is an S-adduct of bisulfite. Additionally, we found four isomeric unstable intermediates
of bisulfite adducted to the heterocyclic ring of Ψ, and their decomposition pathways were
determined. Lastly, the stable Ψ-bisulfite ribose adducts were synthesized in a model RNA
template strand, and reverse transcription was found to generate a deletion at the adduct
consistent with the previous RBS-Seq studies.36 Herein, we present the experiments that led us
to unravel mysteries surrounding this unusual chemistry of high biological relevance.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Results
Interrogation of the reaction between Ψ and bisulfite was first studied in the nucleoside
context. When Ψ was allowed to react with sodium bisulfite following the previous studies
conditions (3 M NaHSO3, pH 5, 16 h, 50 °C; Figure 1A),36 Hypercarb™ HPLC analysis revealed
six product peaks when the reaction was monitored at 220 nm (1-6; Figure 1B). When the reaction
was analyzed at 260 nm, two peaks remained out of the six (1 and 5; Figure 1B). This initial
analysis suggested products 1 and 5 have aromatic components and products 2, 3, 4, and 6 do
not. Each peak was isolated and submitted for ESI--MS analysis to discover that all six products
have the same mass, consistent with a mono-bisulfite adduct (Figure S1). These initial data show
that six isomeric products are formed when Ψ is allowed to reaction with bisulfite. The four
products that are no longer aromatic (2, 3, 4, and 6) will be described in greater detail below
because they were found to be intermediates leading to stable end products. Products 1 and 5
could be the diastereomers described by Everett and Shapiro;17 however, these products elute
under very different mobile phase compositions (A = 20 mM NH4OAc (pH 7) and B = MeOH, 1 =
0% B and 5 = 65% B; Figure 1B), which is not typical of nucleoside diastereomers in our hands
on this HPLC column.37
6
ACS Paragon Plus Environment

Page 7 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. The Ψ-bisulfite reaction yields six products. (A) The reaction conditions leading to (B)
the HPLC analysis monitored at 220 nm or 260 nm to identify those peaks that are no longer
aromatic or retain aromaticity, respectively. The Hypercarb™ HPLC mobile phase composition
consisted of A = 20 mM NH4OAc (pH 7) and B = MeOH, in which 1 eluted with 0% B and 5 eluted
with 65% B.
Prior to structural analysis of 1 and 5, purified samples of each were incubated under
desulfonation conditions (pH 9, 70 °C, 2 h), as well as at pH 5, 7, or 9 at 70 °C for 24 h and both
compounds were recovered without degradation indicating both to be stable bisulfite adducts.
First, product 1 was analyzed by NMR for identification of proton and carbon chemical shifts to
aid in structure determination. The 1H-NMR assignments described are supported by short range
1
1
correlation H,1H-COSY and long-range correlation H,1H-TOCSY NMR analyses that were
particularly helpful for assigning the ribose proton resonances (Figures S2-S4). In compound 1,
one aromatic proton exists at the C6 carbon (7.8 ppm), and signals for each sugar proton were
present. The key peak in the 1H-NMR for 1 was the anomeric proton resonance at 4.4 ppm (Figure
2A); however, it appears as a broad singlet. Comparison of the anomeric proton on 1 with Ψ
provided a modest 0.1 ppm upfield shift. All other ribose protons in 1 were present and provided
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 31
1
2
3
4
5
the anticipated splitting patterns. Noteworthy, the 1H-NMR for 1 was nearly identical to one of the
spectra reported by Everett and Shapiro.17 The 13C-NMR spectrum for 1 was assigned using the
6
7
8
9
1
1
previously determined H resonances and the H,13C-HSQC spectrum (Figures S5 and S6). In
Figure 2C, a plot of the chemical shift changes in 13C signals in 1 relative to Ψ (Δppm = 1 - Ψ)
identifies that the 1‵ carbon resonance was impacted the most, followed by the 2‵ carbon
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
resonance of the sugar, and carbon 5 resonance of the heterocyclic ring. The MS and NMR data
support that compound 1 is a mono-bisulfite adduct at the 1‵ carbon.
Figure 2. NMR data for compounds 1 and 5. The 1H-NMR for (A) 1 and (B) 5 showing the sugar
resonances. The assignments are interpreted with the assistance of H,1H-COSY and H,1H-
TOCSY spectra. Panel C is a plot of the change in 13C signals for 1 and 5 relative to the parent
nucleoside Ψ (Δppm = 1 - Ψ). The assignments were made with the aid of 1H,13C-HSQC spectra.
The numbering scheme for Ψ is provided in the upper left-hand corner of the figure and all NMR
spectra can be found in Figures S2-S11.
1
1
Secondly, product 5 was analyzed by the same NMR techniques as described for
compound 1. The 1H-NMR spectrum for 5 showed one aromatic proton at carbon 6 and all ribose
8
ACS Paragon Plus Environment

Page 9 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
protons (Figures 2B and S7-S9). More interestingly, the 1‵ proton on 5 presented as an expected
doublet that was modestly shifted upfield by 0.2 ppm relative to Ψ. The 13C-NMR peaks for 5
showed the greatest deviation from the parent nucleoside Ψ at the 1‵, 2‵, and 5 carbons (Figures
9
2C, S10, and S11). Additionally, the 1H-NMR spectrum for 5 is very similar to the other spectrum
reported by Everett and Shapiro.17 The MS and NMR data also support compound 5 is a mono-
bisulfite adduct at the 1‵ carbon.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The data to this point might suggest that 1 and 5 are diastereotopic as was suggested by
Everett in his dissertation;17 however, the vastly different HPLC retention profiles led us to
question this assignment (Figure 1B). Attempts to crystallize the purified products failed to
produce diffractable crystals in our hands. Analysis of the IR spectra and fragmentation mass
spectrometry (ESI--MS/MS) spectra for 1 and 5 (Figure S1) confirmed the results obtained from
the NMR data, but added no new insights. Thus, Raman spectroscopy was conducted to
interrogate the nature of the chemical bonds in 1 and 5. Compound 1 provided a Raman spectrum
with a tall peak at 782 cm-1 consistent with a C-S stretch; a similar signal was not present in the
spectrum for 5 (Figure S12). Additionally, a distinctive increase is noted in the S-O stretch at 1035
cm-1, which is characteristic of molecules with an R-SO3 configuration,38 and there is an increase
in the S=O stretch consistent with the presence of the second S=O bond in compound 1.38 Finally,
the strong mode at 980 cm-1 is likely due to S=O symmetric stretching typical of sulfonate
compounds,39 and shows a large increase in intensity typical of the increased polarizability in
compounds containing opposing S=O bonds. These data provide the basis for our assignments
that 1 is a S-adduct of bisulfite at the 1‵ carbon in which the ribose is ring opened, and 5 is an O-
adduct of bisulfite to the 1‵ carbon, also with a ring-opened sugar (Figure 3A).
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 31
1
2
3
4
A
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C
B
ECD Expt. vs. Theory
ECD Expt. vs. Theory
1.4
0.7
0
1.5
1
Compound 1
Calcd (S)-1`-S Adduct
0.5
0
Compound 5
-0.7
-1.4
-0.5
-1
Calcd (R) 1`-O Adduct
190
210
230
250
270
290
310
190
210
230
250
270
290
310
Wavelength (nm)
Wavelength (nm)
Figure 3. Stable ring-opened ribose adducts of bisulfite to Ψ are assigned as S (1) and O (5)
covalent attachments. (A) Scheme illustrating the reaction of bisulfite with Ψ yielding the
constitutional isomer adducts 1 and 5. (B and C) Comparisons of experimental versus theoretical
ECD spectra for 1 and 5 to determine the stereochemistry about the 1‵ carbon in each adduct.
The theoretical spectra were calculated using TD-DFT calculations implemented in the
Gaussian09 program with the M06-2X functional with the 6-311++G(2d,2p) basis set while
implicitly modeling water with the polarizable continuum model. The calculation for the (S) isomer
of the S-adduct was red shifted by 14 nm and the (R) isomer of the O-adduct was red shifted by
22 nm to match the experimental spectra.
Confirmation of the connectivity assignments for 1 and 5 was then supported by a
derivatization experiment. The O-adduct should be susceptible to oxidation of the bisulfite group
to sulfate because of the lone pair on sulfur; such an oxidation would then create a good leaving
group allowing reclosure of the ribose, yielding Ψ or a pyranose isomer depending on whether
the 4‵ or 5‵ OH attacks (Figure 4A). In contrast, the S-adduct should not be prone to oxidation
and should remain underivatized following the oxidation reaction (Figure 4A). The mild oxidant I2
was selected for this test because it readily oxidizes bisulfite but does not damage the heterocyclic
ring complicating the secondary reaction. Treatment of 1 with I2 did not lead to formation of Ψ,
10
ACS Paragon Plus Environment

Page 11 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
while 5 did react with I2 to yield Ψ on the basis of HPLC analysis (Figure 4B). These derivatization
reactions add further support for 1 as the S-adduct and 5 as an O-adduct.
7
8
9
A
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HPLC for Oxidation of 5
HPLC for Oxidation of 1
B
5
Ψ
1
Ψ
1 + I2
5 + I2
Time
Time
Figure 4. Oxidative derivatization supports 1 as an S-adduct and 5 as an O-adduct. (A) Scheme
O vs. S bisulfite oxidation products. (B) Hypercarb™ HPLC analysis showing 5 is susceptible to
oxidative derivatization.
We found it surprising that 1 and 5 eluted as single peaks from the Hypercarb™ HPLC
column, which typically resolves nucleoside diastereomers,37 suggesting 1 and 5 are single
stereoisomers about the 1‵ carbon. This claim is also supported by the NMR data, in which single
peaks for each resonance were observed. Determination of the absolute configurations at the 1
‵ carbons of 1 and 5 was achieved via their electronic circular dichroism (ECD) spectra and
comparison of the experimental and theoretical spectra for each possible isomer of bisulfite
adduction at the 1‵ carbon. The theoretical spectra were calculated using time-dependent density
function theory (TD-DFT) calculations as implemented in the Gaussian09 program with the M02-
6X functional and 6-311++G(2d,2p) basis set while implicitly defining the water solvent with the
polarizable continuum model (PCM). The input structures for the TD-DFT calculations were
geometrically optimized using the B3LYP functional with the same basis set as described for the
TD-DFT calculations.
We have experience applying this approach for stereochemical
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 31
1
2
3
4
5
6
7
8
9
assignments of modified nucleosides.37 The best experimental versus theoretical overlap with 1
was the (S) isomer of the S-adduct (Figures 3B and S13), and the best overlap with 5 was the (R)
isomer of the O-adduct (Figures 3C and S14). Beyond providing experimental evidence for the
stereochemistry about the 1‵ carbon for 1 and 5, these calculations add another level of support
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
for 1 being the S-adduct of bisulfite and 5 being the O-adduct of bisulfite to Ψ (Figure 3A). A final
noteworthy point is that the ECD spectra for 1 and 5 are identical in the direction of light rotation
to those reported by Everett and Shapiro for these two compounds using optical rotary
dispersion.17 Comparison of the spectra identified a few subtle differences in the peak shapes
that can be explained by different experimental approaches and instruments.
Products 2, 3, 4, and 6 that were only detectable at 220 nm have masses consistent with
mono-bisulfite adducts as stated above. Experiments to determine their stabilities were then
conducted. Each intermediate compound was HPLC purified and then subjected to desulfonation
conditions (pH 9, 70 °C, 2 h) followed by HPLC reanalysis to identify the decomposition products.
Compounds 2 and 6 eliminate bisulfite and revert back to Ψ in nearly quantitative yields, while 3
and 4 rearrange to compound 5 also in nearly quantitative yields (>95%; Figure S15). Mild
incubation (pH 7, 22 °C, 24 h) of the compounds (2, 3, 4, and 6) also found decomposition or
rearrangement to the same products in yields >75%. In the RBS-Seq report, a role for Mg2+ to
drive the reaction to products that yield a deletion signature during cDNA synthesis was
suggested. Thus, the intermediate degradation process was monitored in the nucleoside context
with Mg2+ following the RBS-Seq protocol in which a Mg2+ dependency for intermediate
degradation was not observed (Figure S15). The difference in Mg2+ dependency likely results
from the difference in context that exists between the nucleoside studies reported here and the
RNA studies in the previous work. These findings identify compounds 1 and 5 as the final
products of the reaction.
12
ACS Paragon Plus Environment

Page 13 of 31
Journal of the American Chemical Society
1
2
3
4
1
Attempts to characterize compounds 2, 3, 4, and 6 by H- and 13C-NMR were pursued;
5
6
7
8
however, as a result of the instability of these compounds after purification only 2 was successfully
analyzed to yield spectra that were a composite of 2 and its degradation product Ψ (Figures S16
and S17). The known NMR peaks for Ψ40 enabled us to deconvolute the spectra to identify those
peaks occurring from 2. The key diagnostic feature found for 2 was in the 13C-NMR spectra in
which the aromatic carbons C5 and C6 were no longer present and observed at frequencies
associated with sp3 hybridized carbon atoms (73.6 and 66.6 ppm; Figure S17). This supports the
base adduct of bisulfite in 2. The lack of further characterization for 3, 4, and 6 limits our ability
to assign their structures; however, on the basis of known chemistry of bisulfite with pyrimidines,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2,41
the rapid of rate of desulfonation of these products upon heating at pH 7 or greater (Figure
S15), we propose these represent base adducts of bisulfite to Ψ. Consistent with the established
mechanism,2,41 there should be four stereoisomers of bisulfite adducted to the heterocylic ring of
Ψ that was observed with compounds 2, 3, 4, and 6. In conclusion, these results support bisulfite
adduct formation at C6 of the heterocyclic base for all intermediates (Figures 1B, S1, and S16-
S17).
Understanding the structure of the products formed when Ψ was allowed to react with
bisulfite, and how these products decompose and rearrange, led us to probe the reaction in more
detail. First, a time-course analysis in product evolution from 0-240 min was conducted at pH 5
with 3 M NaHSO3 at 50 °C. Product formation was monitored by HPLC at 220 nm, in which it was
assumed that all products have similar extinction coefficients; thus, the peak areas were directly
compared. The time profile found intermediates 4 and 6 were initially formed in highest relative
yield, and as the reaction progressed past 150 min, compound 1 was the major product observed
and compound 5 was detected at approximately one-half the relative concentration of 1 (Figure
5A). The 2:1 relative yield for 1 and 5 was maintained even after a 16 h reaction. Next, the
[NaHSO3] versus yields of 1 and 5 were monitored to find the following trend (Figure 5B). When
13
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 31
1
2
3
4
5
6
7
8
9
the concentration of bisulfite was increased from 0.25 to 2 M the yields of 1 and 5 were nearly
1:1. In contrast, reactions conducted above 2 M bisulfite led to the approximate 2:1 yield of 1 to
5. Additionally, the formation of compound 5, the O-adduct, showed a temperature dependence
with markedly higher yields at higher temperatures compared to compound 1 (Figure 5C). These
observations provide useful insights as described in the discussion section below.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
60%
50%
40%
1
30%
2
6
20%
5
10%
3
4
0%
0
50
100
150
200
250
Time (min)
B
80
70
60
50
40
30
20
10
0
Compd 1 Compd 5
0.25
1
2
3
4
5
[NaHSO3] (M)
C
90
80
70
60
50
40
30
20
10
0
Compd 1
Compd 5
40
50
60
Te mp (°C )
14
ACS Paragon Plus Environment

Page 15 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
Figure 5. (A) Time-dependent product yields for all products, (B) [NaHSO3]-dependent, (C)
temperature-dependent yields for 1 (blue) and 5 (black). The yields were determined by
Hypercarb™ HPLC analysis following the reaction at 220 nm, in which it was assumed that
products 1-6 have the same extinction coefficients.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The ultimate goal of these studies was to understand how the reaction between Ψ and
bisulfite in an RNA strand leads to a deletion signature during cDNA synthesis in RBS-Seq.36
A
24-mer RNA template strand based on one of the control sequences studied in RBS-Seq36 was
prepared by solid-phase synthesis to contain Ψ at a known location for study by reverse
transcription post bisulfite treatment. The RNA oligomer was treated with bisulfite under the
established conditions at pH 5, after which the sample was subjected to desulfonation by
incubating the sample at pH 9 for 2 h at 37 °C. The treated RNA was analyzed by anion-exchange
HPLC separation to determine the conversion to product following the two-step reaction. The
HPLC analysis found a new peak in >90% yield that was purified and verified by ESI--MS to be a
mono-bisulfite-adducted RNA strand (Figure S18). Studies were not pursued to determine the
ratio of products 1 and 5 in the RNA template. Lastly, the RBS-Seq report highlighted an
importance for Mg2+ in generation of the generation signature that appeared to occur during library
preparation; however, the study did not decouple the absolute requirement of this divalent ion for
polymerization and how Mg2+ can impact RNA secondary structures necessary for the deletion
signature. In the present studies, Mg2+ was only added during reverse transcription because
preparation of a sequencing library was not required for the present analysis.
Next, running-start reverse transcription of the template RNA before and after bisulfite
treatment was conducted using SuperScript™ III as the polymerase that we note is a small
change from the SuperScript™ II that was used during RBS-Seq.36 On the basis of the
manufacturer’s notes on the two SuperScript™ products, the key difference is an increase in
thermal stability for III vs. II and for increased cDNA yields from structured RNA templates. The
native RNA template was reverse transcribed to produce a full-length cDNA on the basis of
15
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 31
1
2
3
4
5
6
7
8
9
denaturing polyacrylamide gel electrophoresis (PAGE); in contrast, the HPLC-purified bisulfite-
adducted RNA produced a cDNA one nucleotide shorter (Figure 6). Note that SuperScript™ III
often yields n+1 and n+2 oligomers due to a propensity for blunt-end addition of additional ATPs;42
thus, the bisulfite (-) lanes in Figure 6 show full length (P+7), n+1 (P+8), and n+2 (P+9) bands
while the bisulfite (+) lanes reproduce this pattern exactly but at lengths one nucleotide shorter
(P+6, P+7, and P+8). An additional feature observed in the bisulfite (+) lanes is significant pausing
before the adducted Ψ (P+2) that was not observed in the bisulfite (-) lanes. This final observation
confirms reverse transcription past the Ψ adduct results in a deletion mutation consistent with
published RBS-Seq findings.36 A study of the temperature dependency of reverse transcription
from 35-50 °C found the deletion mutation occurs in high yield with only modest impact of the
temperature at which primer extension was conducted.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Primer extension assay with an RNA template containing Ψ at a known location before
and after bisulfite treatment. (A) The RNA template and DNA primer studied (t = thymidine; the 3`
t increased RNA synthesis yields). (B) Reverse transcription followed by PAGE analysis at
different temperatures (P = unextended primer, and P + number = number of dNTPs
incorporated).
Discussion
16
ACS Paragon Plus Environment

Page 17 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Recently, we assisted the Cairns laboratory to discover RBS-Seq, a bisulfite-based
reaction on RNA that permits sequencing of three RNA modifications simultaneously, including
m1A, m5C, and Ψ.36 The novel observation was that bisulfite treatment of Ψ led to a clear deletion
mutation during cDNA synthesis that provided a sequencing signature to locate this
epitranscriptomic modification. Pseudouridine has long been known to play a role in tRNA and
rRNA tertiary structure,24,43 and its importance is underscored by the fact that 13 pseudouridine
synthetases have been found in humans, four of which are established to operate on mRNA.44
The recent findings that levels of Ψ vary with cellular stress and that high levels of Ψ are formed
in viral RNA have reinvigorated interest in this RNA modification.34,35,45,46
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Sequencing for modifications is best accomplished when two criteria are met: (1) the intact
nucleic acid can be treated with a reagent that reacts quantitatively with a single type of base to
convert it to a different base that can be amplified in an unbiased manner for high-throughput
sequencing. C vs. m5C represents such a case in which the reaction of the bisulfite nucleophile
is at least 50-fold greater with C compared to m5C.4 (2) Ideally, the reagent should react with the
modified base, present in lower abundance, rather than the unmodified base. In this way, a
positive signal is observed rather than seeking the absence of a reaction, which could be due to
any number of factors. C vs.m5C does not meet this criterion, but the reaction remains useful,
particularly for DNA where the strong sequence bias for 5’-CpG sites serves as a double-check
of the presence of m5C. In contrast, the RBS-Seq method positively identifies Ψ compared to U,
because only Ψ provides the deletion signature, and it does so in high yields.36
The goal of the present study was to understand the bisulfite chemistry with Ψ that results
in the deletion signature. We initially addressed this by subjecting the Ψ nucleoside to the
established bisulfite treatment and identified six new products that all had masses consistent with
mono-bisulfite adducts (Figures 1 and S1). Products 1 and 5 retain aromaticity of the heterocycle
giving a strong 260-nm absorbance, while 2, 3, 4, and 6 were only detectable at 220 nm. Stability
17
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 31
1
2
3
4
studies found 1 and 5 to be the stable end products of the reaction that were formed in a 2:1 ratio,
5
6
respectively, under the established RBS-Seq protocol (Figure S15).36
7
8
9
The structures for 1 and 5 were proposed on the basis of complementary 1D- and 2D-
NMR analysis to identify both products were bisulfite adducts at the 1‵ carbon (Figures 2 and S2-
S11). Raman spectroscopy of 1 and 5 provided a key peak for compound 1 at 782 cm-1 that
supports an S-adduct of bisulfite at the 1‵ carbon resulting in a ring-opened sugar. A similar peak
was not observed in the Raman spectrum for 5 leading us to propose an O-adduct of bisulfite at
the 1‵ carbon also resulting in a ring-opened ribose (Figure S12). The O- vs. S-adducts were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
further confirmed by a derivatization reaction (i.e., mild oxidation to the sulfate analog) that yields
a positive result for an O-adduct and a negative result for an S-adduct (Figure 4).
A surprising finding was the observation of a single stereoisomer of each adduct type at
the 1‵ carbon, in which the configurations were assigned by ECD interpreted via TD-DFT
calculations (Figure 3B). Thus, 1 is the (S) isomer of the S-adduct between bisulfite and Ψ with
a ring-opened ribose and 5 is the (R) isomer of the O-adduct between bisulfite and Ψ with a ring-
opened ribose (Figure 3A); both adducts are to the ribose C1’, on the basis of 13C NMR
assignments (Figure 2C). The unusual lack of splitting to the C1’-H of adduct 1 must be
attributable to a rare, but possible, preferred conformation at which the Karplus curve predicts a
coupling constant of 0 Hz (Figure 2A). The proposed structures also provide an explanation for
the different HPLC elution profiles for 1 and 5 that led us to dig deeper in the first place and to
identify the different covalent attachments leading to 1 and 5. Compound 1 is a sulfonic acid
derivative expected to have a low pKa value (< 0), while compound 5 is a sulfinic acid derivative
with a much higher pKa (2-7), and these vastly different acidities likely lead to the different HPLC
retention times observed.
18
ACS Paragon Plus Environment

Page 19 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
On the basis of the structures and the [NaHSO3] dependency in the yields of 1 and 5, a
mechanism is proposed for their formation that addresses the final stereochemistry at the 1‵
7
8
9
carbon of each adduct (Figures 3A and 5B, Scheme 2). Conjugate addition of the nucleophilic
sulfur of bisulfite to C6 of Ψ at pH 5 generates four diastereomeric base adducts, represented by
peaks 2, 3, 4 and 6 in the HPLC chromatogram (Figure 1B); two of the isomers are E and two are
Z. Isomers 2 and 6 must have a trans orientation of C6-sulfite and C5-H, because heating at pH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-
9 leads to elimination of HSO3 to return to starting material Ψ, mostly likely through an E2
mechanism. Overall, this chemistry is essentially identical to the bisulfite sequencing reactions
of C vs. m5C for which pH-reversible conjugate addition to C6 is sterically encumbered for m5C,
and therefore much slower. Hayatsu has previously found anti elimination for bisulfite adducted
T to proceed efficiently, while syn elimination from T required increased heat and alkaline
conditions.41
O
O
O
O
HN
O
NH
HN
R
NH
O-
S
HN
O
NH
HN
R
NH
-O3S
HO
O
O
O
O
-O3S
HO
excess
HSO3
O
S
O
H
H
O
O
-
HO
HN
O
NH
-O
-
+ HSO3
H
H
H
-O
pH 5, 50o
(S)-1
-
O
HSO3
pH 5, 50o
SN2'
HO
H
E2
OH
S
O
HO
HO
O
OH
- HSO3-
pH 9, ?
O
O
-
H
HSO3
O
HO
OH
[2,3]
HN
O
NH
HN
NH
O
HN
NH
-O3S
?
-
SO3
O
pH 9, 70o
O
HN
NH
H
H
O
H
H
HO
O
HO
O
O
S
R
H
O
(R)-5
-O
O
HO
HO
R
-O
S
H
OH
OH
O
2 & 6
(E)
3 & 4
(Z)
Scheme 2. Proposed mechanism of formation of (S)-1 and (R)-5.
The outcome of isomers 3 and 4 is more complex; we propose they have a cis relationship
-
of the C6-SO3 and C5-H groups, and therefore the standard antiperiplanar geometry for an E2
-
elimination of bisulfite (H + SO3 ) cannot be attained. Instead each of these isomers can adopt a
conformation in which the ribose O4’ is antiperiplanar to the acidic hydrogen at C6. Thus, an E2
elimination leads to ribose ring opening of defined stereochemistry (Scheme 2; note, individual
19
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 31
1
2
3
4
5
6
7
8
9
assignments of 2 vs. 6 and 3 vs. 4 could not be made). Peaks 3 and 4 are the smallest
components of the HPLC chromatogram because these products readily undergo further
chemistry, even at pH 5, to produce 1 and 5. We are uncertain at this stage whether peaks 3 and
4 are the initial bisulfite adducts (two E isomers) or their ring-opened isomers. Support for the
ring-opened structures is derived from the UV-vis spectra for 3 and 4 that have greater conjugation
than the ring-closed structures 2 and 6 leading to a small but significant absorption at 260 nm
(Figure S19). In any case, both 3 and 4 can lead to both (S)-1 and to (R)-5 in the following ways:
(1) high concentrations of bisulfite increase the yield of 1 (Figure 5B) that can be formed from
either of the ring-opened 3 or 4 structures by an SN2’ reaction with anti stereochemistry (Scheme
2, blue). The stereochemistry of SN2’ reactions was debated decades ago;47,48 apparently the
reaction surface is sufficiently plastic that the nature of the nucleophile, the leaving group and the
planarity of the intermediate carbon play roles in determining the syn/anti preference. Here we
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-
-
propose an anti orientation of the attacking HSO3 and the leaving HSO3 because these groups
are very bulky, hence disfavoring the syn stereochemistry often observed for SN2’ reactions. (2)
Higher temperatures favor the conversion of both 3 and 4 to compound 5 (Figure 5C), and both
lead to the (R) isomer (Scheme 2, red). This reaction must be a [2,3] sigmatropic shift because
of the carbon-oxygen bond formation at C1’. Only intramolecular attack of an oxygen could create
the C-O bond; any bimolecular nucleophilic attack would create a C-S bond due to the higher
nucleophilicity of sulfur. Finally, we point to the fact that U does not readily undergo reactions
parallel to this, i.e., to ring-opened adducts, because bisulfite adducts to the 5,6-double bond of
U do not place a hydrogen at an appropriate site for E2 ring-opening akin to 3 and 4.
Several features of the mechanistic scheme are noteworthy. First, after the stereorandom
conjugate addition of bisulfite to C6 of Ψ producing four diastereomeric adducts, all subsequent
reactions—E2, SN2’ and the [2,3]-sigmatropic shifts—are stereospecific. The enantiopure
outcomes of (S)-1 and to (R)-5 are directed by the absolute configuration at C-1’ in Ψ. Secondly,
20
ACS Paragon Plus Environment

Page 21 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
while bisulfite addition/elimination reactions have been well studied with cytidine, the SN2’ reaction
in Scheme 2 is unprecedented. The driving force for this reaction in the forward direction must
be the rearomatization of the uracil ring. Thirdly, we are unaware of any reports of [2,3]
sigmatropic shifts involving a sulfonate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The final step of our studies was to treat a synthetic RNA strand containing a single Ψ at
a known site with bisulfite followed by desulfonation to generate the adducts 1 and 5. The HPLC-
purified, adducted strands were used as templates for reverse transcription to discover a deletion
signature when the adduct was present (Figure 6). A prior study by the Grollman laboratory found
that when a DNA polymerase traverses a ring-opened sugar in a DNA template strand, the
damaged site is extruded, and polymerization continues yielding a deletion mutation.49
Apparently, the ribose ring-opened adducts of bisulfite to Ψ (1 and 5) in an RNA template also
result in extrusion during reverse transcription leading to a deletion signature, on the basis of the
present findings. The deletion signature yield observed during reverse transcription was nearly
quantitative, while the deletion signature found during DNA polymerization was modest
suggesting that both DNA vs. RNA context and the nature of the polymerase have an impact on
the yield. The critical details differentiating the deletion yield between the two studies is not known
at present; although, reverse transcriptases bypass modified nucleotides with higher efficiency
albeit with lower fidelity.50 A final study to ensure that the ribose adducts did not simply change
the acid-base properties or hydrogen bonding potential of Ψ to cause the deletion signature was
ruled out via an acid-base titration study of 1 and 5. The titrations were followed by UV-vis to find
that the pKa values for the heterocyclic rings of 1 and 5 were within error of the measured pKa for
Ψ at 9.5 (Figure S20). This final study aids in the claim that opening the furanose ring of Ψ via
bisulfite adduction leads to the deletion signature during cDNA synthesis.
These deletions are conveniently identified during next-generation sequencing during the
RBS-Seq procedure for identification of sites in the transcriptome in which a U was isomerized to
21
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 31
1
2
3
4
5
6
7
8
9
Ψ.36 The key advantage provided by read–through of the adducted Ψ is that more than one
modification per template strand can be mapped. This approach thus demonstrates a significant
advantage over other methods for locating Ψ by, for example, introduction of polymerase stops
using CMC prior to cDNA synthesis.31-35 Identification of RNA modifications by a stop in cDNA
synthesis is notorious for yielding false positives from reverse transcription stops resulting from
other features of the RNA. Additionally, only one Ψ per strand can be detected by the CMC
approach.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
The discovery that the epitranscriptomic modifications m1A, m5C, and Ψ can be mapped
by bisulfite treatment via RBS-Seq provides a significant advance in sequencing for these critical
modifications.36 Renewed interest in Ψ, due to better experimental approaches to its detection,
is leading to new insights into the role of this modification as a modulator of RNA function and as
a response to cellular stress.34,35,45,46 During RBS-Seq, Ψ is located via a deletion signature that
occurs after bisulfite treatment. Herein, we applied the bisulfite reaction to Ψ, first in the
nucleoside context to monitor the reaction in which six isomeric mono-bisulfite products were
formed. Two of these products, 1 and 5, are stable end products with bisulfite adducted at the 1‵
carbon, resulting in opening of the sugar. Prior work reported in a Ph.D. dissertation from the
Shapiro laboratory studied the bisulfite addition to Ψ and proposed a pair of diastereomers being
formed with S-adduction at the 1‵ carbon;17 however, puzzling data, such as the unsplit C1’ proton
of compound 1 must have discouraged them from publication of the results. Using modern
spectroscopic techniques coupled with classical product derivatization, we found that the two
products formed are constitutional isomers in which 1 is the (S) isomer of the S-adduct of bisulfite
at the 1‵ carbon and 5 is the (R) isomer of the O-adduct at the same carbon (Figure 3A). These
products are highly stable and formed in a ratio of 2:1 for 1 and 5, respectively. Several
noteworthy aspects of the reaction mechanism include the series of stereoselective reactions—
22
ACS Paragon Plus Environment

Page 23 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
E2, SN2’ and a [2,3]-sigmatropic shift—with the latter two being unprecedented in the bisulfite
literature (Scheme 2).
7
8
9
In a second part of the study, we treated a synthetic RNA template with bisulfite to
generate 1 and 5 at a known location to then find that a deletion occurs after reverse transcription.
This final observation in tandem with all the other studies identifies the source of the deletion
signature at Ψ that occurs when RBS-Seq is applied to the transcriptome.36 The ability to locate
the epitransciptomic modification Ψ by a deletion signature during cDNA synthesis permits
location of more than one modification per template strand that is a significant advance over prior
methods.31-35 The initial observation of bisulfite treatment of dC/C to effect deamination to dU/U,
while the same reaction of 5mC/m5C proceeds with 50x slower kinetics has been harnessed by
researchers to sequence these epigenomic/epitranscriptomic modifications via high-throughput
sequencing. Application of this technique has shed light on many aspects of chemical
modification of DNA and RNA to impact cellular phenotype. This classical reaction, once
harnessed to facilitate recrystallization of aldehydes and ketones, has revolutionized our
understanding of biology and now deserves reintroduction to students of organic chemistry.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methods
Nucleoside reaction and product characterization. The Ψ nucleoside is commercially
available, as well as all other reagents that were used without further purification. The Ψ
nucleoside reactions with bisulfite were conducted following the RBS-Seq protocol.36 In a reaction
volume of 1 mL was added 1 mM Ψ and 3 M freshly prepared NaHSO3 at pH 5.0. The reaction
mixture was placed in a 50 °C heat block to react for 16 h, after which the solution was removed
from the heat and directly analyzed by HPLC using a Hypercarb™ column running A = 20 mM
NH4OAc (pH 7) and B = MeOH. The HPLC method consisted of a flow rate of 1 mL/min holding
an isocratic gradient of 0% B for the first 5 min followed by a linear increase in B to 100% over 10
min that was held for 15 min. The HPLC analysis indicated six product peaks of which 1 and 5
23
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 31
1
2
3
4
5
6
7
8
9
were the stable end products and were fully characterized and the others were unstable
intermediates characterized by mass spectrometry. 1. tR = 5 min. HRMS (ESI--TOF) calcd for
C9H13N2O9S [M-H]- = 325.0342; found 325.0349. ESI--TOF-MS/MS parent = 325.0349, daughters
= 204.9920, 183.0407, 161.9861, 153.0299, 141.0299, 140.0345, 123.0196, 110.0243, 98.0244,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
80.9651, and 79.9573. H-NMR (500 MHz, D2O) δ 7.76 (s, 1 H), 4.40 (s, J = 0 Hz, 1 H), 4.31 (dd,
J = 8.32 Hz, 1 H), 3.67 (m, J = 2.45 Hz, 1 H), 3.61 (d, J = 2.17 Hz, 1 H), 3.45 (d, J = 6.85 Hz, 1
H), 3.22 (m, J = 4.89 Hz, 1 H). 13C-NMR (125 MHz, D2O) δ 168.6, 155.0, 146.1, 107.7, 75.1, 74.9,
73.0, 64.5, 58.1. UV-vis in ddH2O λmax = 265 nm (ε = 6,000 L•mol-1•cm-1); ECD in ddH2O λ nm (c
= 2 x 10-4 M in ddH2O) 266 (Δε = +14.4) and 212 (Δε = -40.3). Raman shift cm-1 (ddH2O) 440,
550, 650, 775, 900, 950, 1050, 1400, 1625, 2900. 5. tR = 12.5 min. HRMS (ESI--TOF) calcd for
C9H14N2O9S [M - H]- = 325.0342; found 325.0341. ESI--TOF-MS/MS parent = 325.0341,
daughters = 204.9917, 183.0396, 161.9855, 153.0298, 141.0296, 140.0344, 123.0192, 110.0240,
1
98.0242, and 80.9652. Intermediates. H-NMR (500 MHz, D2O) δ 7.61 (s, 1 H), 4.39 (d, J = 6.85
Hz, 1 H), 3.98 (dd, J = 6.85 Hz, 1 H), 3.75 (dd, J = 5.87 Hz, 1 H), 3.65 (m, J = 3.41 Hz, 1 H), 3.58
(d, J = 3.22 Hz, 1 H), 3.44 (d, J = 6.85 Hz, 1 H). 13C-NMR (125 MHz, D2O) δ 184.0, 167.9, 155.0,
144.7, 110.8, 75.0, 74.2, 64.9, 58.8. UV-vis in ddH2O λmax = 266 nm (ε = 6,200 L•mol-1•cm-1); ECD
in ddH2O λ nm (c = 1 x 10-4 M in ddH2O) 268 (Δε = -8) and 211 (Δε = +22). Raman shift cm-1
(ddH2O) 550, 775, 900, 950, 1050, 1625, 2900. 2. tR = 10 min (ESI--MS) calcd [M-H]- = 325.27;
found 325.00. 1H-NMR (500 MHz, D2O) δ 4.43 (s, 1 H), 3.97 (m, J = 4.89 Hz, 1 H), 3.89 (m, J =
5.87 Hz, 1 H), 3.79 (m, J = 4.89 Hz, 1 H), 3.46 (dd, J = 5.38 Hz, 1 H). 13C-NMR (125 MHz, D2O)
δ 173.1, 143.9, 86.7, 82.6, 75.0, 73.6, 73.2, 66.6, 64.1. 3. tR = 10.5 min (ESI--MS) calcd [M-H]- =
325.27; found 325.07. 4. tR = 11.5 min (ESI--MS) calcd [M-H]- = 325.27; found 325.07; 6. tR = 13
min (ESI--MS) calcd [M-H]- = 325.27; found 325.18.
Density functional theory calculations. All calculations were performed using DFT
methods as implemented in the Gaussian09 package, and visualized using GaussView (v5).52
24
ACS Paragon Plus Environment

Page 25 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
The 6-311++G(2d,2p) basis set,53,54 B3LYP55,56 or M06-2X functional,57 and the polarizable
continuum model (PCM)58,59 for implicit definition of water were used for the calculations. The
computed ECD spectra were completed using TD-DFT theory to calculate 25 excitations.60-62
Complete details for plotting the theoretical spectra has previously been reported by our
laboratory.37
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Polymerase bypass assay. The RNA template and DNA primer were synthesized by
solid-phase synthesis following established protocols using commercially available
phosphoramidites (RNA = 5‵-UA CUΨ CAA GGU GGA AGU UAG AGG t-3‵; and DNA = 5‵-CCT
CTA ACT TCC ACC T-3‵). The RNA strand was synthesized with a 3‵ thymidine (t) to increase
the synthesis yield, and it does not impact the reverse transcription reaction. The Ψ-containing
RNA strand was subjected to the bisulfite reaction as described for the nucleoside, with one
change. After the 16 h reaction, the NaHSO3 was removed via a NAP-5 column, after which the
RNA was desulfonated by heating at 37 °C in 1 M Tris buffer (pH 9) for 2 h. Upon desulfonation,
the RNA was immediately purified by anion-exchange HPLC using the mobile phases A = 1:9
MeCN:ddH2O and B = 1.5 M NaOAc (pH 7) in 1:9 MeCN:ddH2O. The HPLC method was initiated
at 25% B and increased linearly to 100% B over 30 min with a 1 mL/min flow rate while monitoring
the elution via absorbance at 260 nm. The HPLC-purified adducted RNA strands were
characterized by ESI--MS: calcd mass = 7854.7; found 7854.1. In brief, the reverse transcription
experiments were conducted by first placing a 5‵ 32P on the end of the DNA for visualization of
the polymerization reaction by denaturing PAGE. Next, a reaction with 10-μL volume containing
100 nM pre-annealed RNA template and DNA primer duplex, 3 mM MgCl2, 10 mM DTT, and 500
μM of each dNTP was prepared. The extension reaction was thermally equilibrated at the desired
reaction temperature for 15 min prior to addition of 10 U of SuperScript™ III. The reaction was
allowed to progress for 40 min followed by heat denaturation to quench the reaction prior to
analysis by PAGE. Complete experimental details are provided in the Supporting Information.
25
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 31
1
2
3
4
5
6
Supporting Information
Supporting Information is available.
7
8
9
Complete experimental details, MS, NMR, HPLC, IR, Raman spectra, UV-vis, and DFT
optimized geometries and energies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conflict of Interest
The authors are not in conflict of interest in this work.
ORCID
Cynthia J. Burrows: 0000-0001-7523-8529
Aaron M. Fleming: 0000-0002-2000-0310
Joel M. Harris: 0000-0002-7081-8188
Jay P. Kitt: 0000-0002-1469-3659
Acknowledgments
Financial support of this research was provided by the NIH via grant no. R01 GM093099
to CJB and the NSF via grant no. CHE 1904424 to JMH. The DNA and RNA strands were
synthesized by the University of Utah Health Sciences Core facilities that are supported in part
by a National Cancer Center Support grant (P30 CA042014). The support and resources from
the Center for High Performance Computing at the University of Utah are gratefully
acknowledged. We are grateful to Prof. Jon Rainier (Univ. of Utah) for helpful discussions. The
work presented herein was inspired by the astute observations of Dr. Vahid Khoddami and Prof.
Bradley Cairns at the University of Utah while developing RBS-Seq.
References
26
ACS Paragon Plus Environment

Page 27 of 31
Journal of the American Chemical Society
1
2
3
4
5
(1)
Morrison, R. T.; Boyd, R. N.: Organic Chemistry; Allyn and Bacon, Inc.: Boston,
MA, USA, 1973; Vol. 3rd.
6
7
8
(2)
Shapiro, R.; Servis, R. E.; Welcher, M. Reactions of uracil and cytosine derivatives
with sodium bisulfite. J. Am. Chem. Soc. 1970, 92, 422-424.
9
(3)
Hayatsu, H.; Wataya, Y.; Kai, K.; Iida, S. Reaction of sodium bisulfite with uracil,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cytosine, and their derivatives. Biochemistry 1970, 9, 2858-2865.
(4)
Wang, R. Y.; Gehrke, C. W.; Ehrlich, M. Comparison of bisulfite modification of 5-
methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 1980, 8, 4777-4790.
(5)
Frommer, M.; McDonald, L. E.; Millar, D. S.; Collis, C. M.; Watt, F.; Grigg, G. W.;
Molloy, P. L.; Paul, C. L. A genomic sequencing protocol that yields a positive display of 5-
methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 1827-
1831.
(6)
Booth, M. J.; Raiber, E.-A.; Balasubramanian, S. Chemical methods for decoding
cytosine modifications in DNA. Chem. Rev. 2015, 115, 2240-2254.
(7)
Song, C. X.; Yi, C.; He, C. Mapping recently identified nucleotide variants in the
genome and transcriptome. Nat. Biotechnol. 2012, 30, 1107-1116.
(8)
Booth, M. J.; Branco, M. R.; Ficz, G.; Oxley, D.; Krueger, F.; Reik, W.;
Balasubramanian, S. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine
at single-base resolution. Science 2012, 336, 934-937.
(9)
Yu, M.; Hon, G. C.; Szulwach, K. E.; Song, C. X.; Zhang, L.; Kim, A.; Li, X.; Dai,
Q.; Shen, Y.; Park, B.; Min, J. H.; Jin, P.; Ren, B.; He, C. Base-resolution analysis of 5-
hydroxymethylcytosine in the mammalian genome. Cell 2012, 149, 1368-1380.
(10) Song, C. X.; Szulwach, K. E.; Dai, Q.; Fu, Y.; Mao, S. Q.; Lin, L.; Street, C.; Li, Y.;
Poidevin, M.; Wu, H.; Gao, J.; Liu, P.; Li, L.; Xu, G. L.; Jin, P.; He, C. Genome-wide profiling of 5-
formylcytosine reveals its roles in epigenetic priming. Cell 2013, 153, 678-691.
(11) Booth, M. J.; Marsico, G.; Bachman, M.; Beraldi, D.; Balasubramanian, S.
Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat. Chem. 2014,
6, 435-440.
(12) Lu, X.; Song, C. X.; Szulwach, K.; Wang, Z.; Weidenbacher, P.; Jin, P.; He, C.
Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection
in DNA. J. Am. Chem. Soc. 2013, 135, 9315-9317.
(13) Wang, Y.; Zhang, X.; Zou, G.; Peng, S.; Liu, C.; Zhou, X. Detection and application
of 5-formylcytosine and 5-formyluracil in DNA. Acc. Chem. Res. 2019, 52, 1016-1024.
(14) Dietzsch, J.; Feineis, D.; Hobartner, C. Chemoselective labeling and site-specific
mapping of 5-formylcytosine as a cellular nucleic acid modification. FEBS Lett. 2018, 592, 2032-
2047.
27
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 31
1
2
3
4
5
6
(15) Carell, T.; Kurz, M. Q.; Muller, M.; Rossa, M.; Spada, F. Non-canonical bases in
the genome: The regulatory information layer in DNA. Angew. Chem. Int. Ed. Engl. 2018, 57,
4296-4312.
7
8
9
(16) Singhal, R. P. Chemical probe of structure and function of transfer ribonucleic
acids. Biochemistry 1974, 13, 2924-2932.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17) Everett, D. W. Part i: reaction of pseudouridine with bisulfite. part ii: reaction of
glyoxal with guanine derivatives: a spectrophotometric probe of molecular structure. Ph.D.
Dissertation, New York University, 1980.
(18) Nachtergaele, S.; He, C. Chemical modifications in the life of an mRNA transcript.
Annu. Rev. Genet. 2018, 52, 349-372.
(19) Meyer, K. D.; Jaffrey, S. R. Rethinking m(6)A readers, writers, and erasers. Annu.
Rev. Cell Dev. Biol. 2017, 33, 319-342.
(20) Huber, S. M.; Leonardi, A.; Dedon, P. C.; Begley, T. J. The versatile roles of the
tRNA epitranscriptome during cellular responses to toxic exposures and environmental stress.
Toxics 2019, 7.
(21) Martinez, N. M.; Gilbert, W. V. Pre-mRNA modifications and their role in nuclear
processing. Quant. Biol. 2018, 6, 210-227.
(22) Roundtree, I. A.; Evans, M. E.; Pan, T.; He, C. Dynamic RNA modifications in gene
expression regulation. Cell 2017, 169, 1187-1200.
(23) Song, J.; Yi, C. Chemical modifications to RNA: A new layer of gene expression
regulation. ACS Chem. Biol. 2017, 12, 316-325.
(24) Jiang, J.; Seo, H.; Chow, C. S. Post-transcriptional modifications modulate rRNA
structure and ligand interactions. Acc. Chem. Res. 2016, 49, 893-901.
(25) Fisher, A. J.; Beal, P. A. Structural basis for eukaryotic mRNA modification. Curr.
Opin. Struct. Biol. 2018, 53, 59-68.
(26) Harcourt, E. M.; Kietrys, A. M.; Kool, E. T. Chemical and structural effects of base
modifications in messenger RNA. Nature 2017, 541, 339-346.
(27) Grozhik, A. V.; Jaffrey, S. R. Distinguishing RNA modifications from noise in
epitranscriptome maps. Nat. Chem. Biol. 2018, 14, 215-225.
(28) Morse, D. P.; Bass, B. L. Detection of inosine in messenger RNA by inosine-
specific cleavage. Biochemistry 1997, 36, 8429-8434.
(29) Hafner, M.; Landthaler, M.; Burger, L.; Khorshid, M.; Hausser, J.; Berninger, P.;
Rothballer, A.; Ascano, M.; Jungkamp, A.-C.; Munschauer, M.; Ulrich, A.; Wardle, G. S.; Dewell,
S.; Zavolan, M.; Tuschl, T. Transcriptome-wide identification of RNA-binding protein and
microRNA target sites by PAR-CLIP. Cell 2010, 141, 129-141.
28
ACS Paragon Plus Environment

Page 29 of 31
Journal of the American Chemical Society
1
2
3
4
5
6
(30) Squires, J. E.; Patel, H. R.; Nousch, M.; Sibbritt, T.; Humphreys, D. T.; Parker, B.
J.; Suter, C. M.; Preiss, T. Widespread occurrence of 5-methylcytosine in human coding and non-
coding RNA. Nucleic Acids Res. 2012, 40, 5023-5033.
7
8
9
(31) Carlile, T. M.; Rojas-Duran, M. F.; Zinshteyn, B.; Shin, H.; Bartoli, K. M.; Gilbert,
W. V. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human
cells. Nature 2014, 515, 143-146.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(32) Li, X.; Zhu, P.; Ma, S.; Song, J.; Bai, J.; Sun, F.; Yi, C. Chemical pulldown reveals
dynamic pseudouridylation of the mammalian transcriptome. Nat. Chem. Biol. 2015, 11, 592-597.
(33) Lei, Z.; Yi, C. A radiolabeling-free, qPCR-based method for locus-specific
pseudouridine detection. Angew. Chem. Int. Ed. Engl. 2017, 56, 14878-14882.
(34) Schwartz, S.; Bernstein, D. A.; Mumbach, M. R.; Jovanovic, M.; Herbst, R. H.;
Leon-Ricardo, B. X.; Engreitz, J. M.; Guttman, M.; Satija, R.; Lander, E. S.; Fink, G.; Regev, A.
Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA
and mRNA. Cell 2014, 159, 148-162.
(35) Lovejoy, A. F.; Riordan, D. P.; Brown, P. O. Transcriptome-wide mapping of
pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae. PloS One
2014, 9, e110799.
(36) Khoddami, V.; Yerra, A.; Mosbruger, T. L.; Fleming, A. M.; Burrows, C. J.; Cairns,
B. R. Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base
resolution. Proc. Nat. Acad. Sci. U.S.A. 2019, 116, 6784-6789.
(37) Fleming, A. M.; Orendt, A. M.; He, Y.; Zhu, J.; Dukor, R. K.; Burrows, C. J.
Reconciliation of chemical, enzymatic, spectroscopic and computational data to assign the
absolute configuration of the DNA base lesion spiroiminodihydantoin. J. Am. Chem. Soc. 2013,
135, 18191-18204.
(38) Houlton, H. G.; Tartar, H. V. Raman spectra of sodium alkyl sulfonates and
sulfinates. J. Am. Chem. Soc. 1938, 60, 544-548.
(39) Ben Mabrouk, K.; Kauffmann, T. H.; Aroui, H.; Fontana, M. D. Raman study of
cation effect on sulfate vibration modes in solid state and in aqueous solutions. J. Raman
Spectrosc. 2013, 44, 1603-1608.
(40) Wishart, D. S.; Knox, C.; Guo, A. C.; Eisner, R.; Young, N.; Gautam, B.; Hau, D.
D.; Psychogios, N.; Dong, E.; Bouatra, S.; Mandal, R.; Sinelnikov, I.; Xia, J.; Jia, L.; Cruz, J. A.;
Lim, E.; Sobsey, C. A.; Shrivastava, S.; Huang, P.; Liu, P.; Fang, L.; Peng, J.; Fradette, R.; Cheng,
D.; Tzur, D.; Clements, M.; Lewis, A.; De Souza, A.; Zuniga, A.; Dawe, M.; Xiong, Y.; Clive, D.;
Greiner, R.; Nazyrova, A.; Shaykhutdinov, R.; Li, L.; Vogel, H. J.; Forsythe, I. HMDB: a
knowledgebase for the human metabolome. Nucleic Acids Res. 2009, 37, D603-D610.
(41) Hayatsu, H. Discovery of bisulfite-mediated cytosine conversion to uracil, the key
reaction for DNA methylation analysis--a personal account. Proc. Jpn. Acad. Ser. B Phys. Biol.
Sci. 2008, 84, 321-330.
29
ACS Paragon Plus Environment