Product and mechanistic analysis of the reactivity of a C6-pyrimidine radical in RNA

Article abstract of DOI:10.1021/ja200317w

Nucleobase radicals are the major reactive intermediates produced when hydroxyl radical reacts with nucleic acids. 5,6-Dihydrouridin-6-yl radical (1) was independently generated from a ketone precursor via Norrish Type I photocleavage in a dinucleotide, single-stranded, and double-stranded RNA. This radical is a model of the major hydroxyl radical adduct of uridine. Tandem lesions resulting from addition of the peroxyl radical derived from 1 to the 5′-adjacent nucleotide are observed by ESI-MS. Radical 1 produces direct strand breaks at the 5′-adjacent nucleotide and at the initial site of generation. The preference for cleavage at these two positions depends upon the secondary structure of the RNA and whether O₂ is present or not. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. In general, strand scission is significantly more efficient under anaerobic conditions than when O₂ is present. Strand scission is more than twice as efficient in double-stranded RNA than in a single-stranded oligonucleotide under anaerobic conditions. Internucleotidyl strand scission occurs via β-fragmentation following C2′-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical ultimately yields products containing 3′-phosphate or 3′-deoxy-2′-ketouridine termini. These end groups are proposed to result from competing deprotonation pathways. The dependence of strand scission efficiency from 1 on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions.

Full text of DOI:10.1021/ja200317w

ARTICLE
pubs.acs.org/JACS
Product and Mechanistic Analysis of the Reactivity of a C6-Pyrimidine
Radical in RNA
Aaron C. Jacobs,† Marino J. E. Resendiz,† and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S Supporting Information
b
ABSTRACT: Nucleobase radicals are the major reactive inter-
mediates produced when hydroxyl radical reacts with nucleic
acids. 5,6-Dihydrouridin-6-yl radical (1) was independently
generated from a ketone precursor via Norrish Type I photo-
cleavage in a dinucleotide, single-stranded, and double-stranded
RNA. This radical is a model of the major hydroxyl radical
adduct of uridine. Tandem lesions resulting from addition of the
peroxyl radical derived from 1 to the 50-adjacent nucleotide are
observed by ESI-MS. Radical 1 produces direct strand breaks
at the 50-adjacent nucleotide and at the initial site of generation. The preference for cleavage at these two positions depends upon the
secondary structure of the RNA and whether O2 is present or not. Varying the identity of the 50-adjacent nucleotide has little effect
on strand scission. In general, strand scission is significantly more efficient under anaerobic conditions than when O2 is present.
Strand scission is more than twice as efficient in double-stranded RNA than in a single-stranded oligonucleotide under anaerobic
conditions. Internucleotidyl strand scission occurs via β-fragmentation following C20-hydrogen atom abstraction by 1. The
subsequently formed olefin cation radical ultimately yields products containing 30-phosphate or 30-deoxy-20-ketouridine termini.
These end groups are proposed to result from competing deprotonation pathways. The dependence of strand scission efficiency
from 1 on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA
structural information from hydroxyl radical reactions.
’ INTRODUCTION
Nucleobase radicals constitute the major family of reactive
species produced in nucleic acids that are exposed to hydroxyl
radical (OH ), which is produced by γ-radiolysis and metal
3
complexes (e.g., Fe EDTA). Consequently, the chemical fates of
3
nucleobase radicals are significant when determining the effects
of these damaging agents. For many years, RNA was used as a
substrate for studying the chemistry of γ-radiolysis and signifi-
cant advances in our understanding of nucleic acid radical
reactivity were achieved by combining a variety of analytical
techniques with random generation of reactive species by ioniz-
ing radiation within the biopolymer.1,2 Although DNA damage
chemistry was also studied in this way, elucidation of the
reactivity of radicals formed in this biopolymer has benefited
from an approach in which reactive intermediates and metastable
lesions are independently generated from modified nucleotides
at specific sites within synthetic biopolymers.3-14 Synthetically
controlled formation of reactive intermediates and DNA lesions
has helped resolve mechanistic controversies,7,10 and uncover
novel reaction pathways.11,12 Independent RNA radical genera-
tion from modified nucleotides to produce reactive intermediates
has been employed less frequently.6,15 However, the increasing
Recently, we reported on the formation of direct strand breaks
from 5,6-dihydrouridin-6-yl radical (1) following its photochemical
generation from 2 (eq 1).22 Norrish type I cleavage has been used
to generate several nucleic acid radicals.5,13,14,23 The ketone (2)
produces 5,6-dihydrouridin-6-yl radical (1) along with an equiva-
lent of t-butyl radical and carbon monoxide via type I cleavage.
Decarbonylation of the acyl radical intermediate (g105 s-1) is
rapid and the diffusible t-butyl radical is produced in subnanomolar
use of OH cleavage to probe RNA structure and folding
3
kinetics16-19 and the importance of RNA damage in diseases
have provided greater impetus to further unravel the underlying
chemistry of its oxidative damage.20,21
Received: January 12, 2011
Published: March 10, 2011
r
2011 American Chemical Society
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Scheme 1
would be an exception to the generally accepted oxygen enhance-
ment effect.1 Such consequences prompted us to more comple-
tely examine the reactivity of 1 in RNA.
’ RESULTS AND DISCUSSION
Product Analysis from 5,6-Dihydrouridin-6-yl (1) Flanked
by Uridine in Duplex RNA under Anaerobic Conditions.
Initial studies showed that direct strand breaks were produced
most efficiently when 5,6-dihydrouridin-6-yl (1) was generated
in duplex RNA under anaerobic conditions.22 Denaturing poly-
acrylamide gel electrophoresis (PAGE) analysis coupled with
enzymic end group analysis of photolyzed 30-32P-7 (please note
that the radical-containing strand is radiolabeled in all experi-
ments) revealed two products each containing 50-phosphate
groups. Comparison with independently synthesized oligonu-
cleotide markers showed that the major and minor products
resulted from oxidation at the 50-adjacent (internucleotidyl) and
nucleobase radical containing (intranucleotidyl) nucleotides,
respectively. Similar analysis of 50-32P-7 identified the corre-
sponding fragments containing 30-phosphate termini, as well as a
product containing a ribose fragment that resulted from inter-
nucleotidyl oxidation.22 MALDI-TOF MS analysis of the crude
photolysate of 7 confirmed the formation of the 4 phosphory-
lated fragmentation products (12-15, Figure 1). Oligonucleo-
tide 12 corresponds to the major product observed by gel
electrophoresis when 50-32P-7 is photolyzed under anaerobic
conditions. It results from internucleotidyl hydrogen atom
abstraction by 1 as discussed below (Scheme 2). Similarly, 14
corresponds to the 30-phosphate product resulting from intra-
nucleotidyl hydrogen atom abstraction.
concentration.24 5,6-Dihydrouridin-6-yl radical (1) was chosen as a
synthetically expedient analogue of the major OH adduct of
3
uridine (3).25 Addition to the C5-position of uridine accounts
for ∼70% of the reactions between OH andthe pyrimidineringin
3
poly(U).2 Direct strand breaks from 1 were produced at the 50-
adjacent nucleotide (internucleotidyl) and at the nucleotide where
the radical was originally produced (intranucleotidyl). Strand
scission was most efficient in double-stranded RNA under anae-
robic conditions and was reduced significantly in the presence of
O2. Under anaerobic conditions, direct strand scission was more
than 2-fold greater when 1 was flanked by uridines in a duplex
compared to a single-stranded oligonucleotide. This RNA nucleo-
base radical reactivity was distinct from a similar DNA radical (4),
which does not yield significant amounts of direct strand scission
under either of the above conditions regardless of secondary
structure.12,26,27 Isotopic labeling and product studies on a dinu-
cleotide in which 1 was produced indicated internucleotidyl strand
scission followed C20-hydrogen atom abstraction (5, Scheme 1).
Subsequent cleavage by β-elimination from 5 yields radical cation
6, consistent with observations in DNA.5,28 Internucleotidyl strand
scission via C20-hydrogen atom abstraction by 1 and not 4 was
ascribed to the significant reduction in the bond dissociation energy
of the corresponding carbon-hydrogen bond in RNA compared
to DNA.29
Observations reported in the above study indicate that oxida-
tive RNA damage may have physiological consequences and
possible utility for studying biopolymer structure and folding.
RNA structure determination by OH cleavage is typically carried
3
out under aerobic conditions. If this chemistry occurs in a variety
of sequences, the preliminary results summarized above suggest
that it may be possible to extractmore structural information from
such experiments by comparing cleavage patterns under aerobic
and anaerobic conditions. Enhanced cleavage at a particular site in
the absence of O2 relative to that observed under aerobic
conditions will indicate duplex structure. In addition, if C6-
pyrimidine nucleobase radicals account for ∼70% of the reactions
Oligonucleotides, 13 and 15 correspond to the 30-fragments
containing 50-phosphates that are observed by gel electrophor-
esis when 30-32P-7 is photolyzed. An additional ion (m/z =
2835.4) corresponding to 11a (or another species containing the
same m/z) was also observed. Treatment of crude 50-32P-7
between OH and uridine, the preliminary data suggest that RNA
3
may be more susceptible to cleavage in O2 deficient cells. This
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Figure 1. MALDI-TOF MS analysis of crude photolysate of 7.
Table 1. Direct Strand Scission under Anaerobic Conditions from 5,6-Dihydrouridin-6-yl (1) Flanked by Adenosine
absolute yield (%):
substratea
inter.
intra.
phosphate
ribose frag.
phosphate
ribose frag.
total
% inter.
ds (8)
27.7 ( 4.5
5.1 ( 0.6
4.9 ( 1.0
3.2 ( 0.6
4.9 ( 0.9
4.1 ( 0.1
-
38.3 ( 3.3
15.7 ( 0.4
85.8 ( 4.4
52.4 ( 2.2
ss (10)
3.3 ( 0.4
a The substrate is designated by its secondary structure (ss, ds).
photolysate with NaBH4 resulted in a mobility shift in 11a,
consistent with its assignment as a ketone.30 Although other
products with this molecular weight would react similarly with
NaBH4, mechanistic considerations (vide infra) lead us to
propose that 11a is the internucleotidyl cleavage product con-
taining a ribose fragment.
3.7%) within a duplex. In addition, the ratio of internucleotidyl
cleavage products containing 30-phosphate termini relative to
those containing the 30-ribose fragment (11a,b) are also within
experimental error of one another (U1U = 5.8 ( 1.2; A1A = 5.9 (
2.4). The relative amount of internucleotidyl and intranucleo-
tidyl strand scission products in single stranded substrates was
the most significant difference in products formed from 1 flanked
by A (10) or U (9). The original report on 5,6-dihydrouridin-6-yl
radical (1) reactivity showed that intranucleotidyl strand scission
accounted for more than 80% of direct strand breaks when 1 is
flanked by uridines in a single-stranded substrate.22 In contrast,
internucleotidyl oxidation accounts for ∼50% of the direct strand
breaks in single-stranded RNA when 1 is flanked by adenosines
(Table 1). Despite this change in the proportion of cleavage
products in single-stranded RNA (ssRNA), the overall prefer-
ence for direct strand scission when 1 is produced within a duplex
(dsRNA) compared to ssRNA was very similar in the two
flanking sequences (dsRNA/ssRNA: U1U = 2.2, A1A = 2.4).
5,6-Dihydrouridin-6-yl Radical (1) Reactivity Flanked by
Uridine under Aerobic Conditions. Direct strand scission is
significantly reduced under aerobic conditions.22 O2 is expected
to react with 1 and any subsequent alkyl radical formed much
faster (k ∼ 2 ꢀ 109 M-1 s-1, [O2] ∼ 0.2 mM) than these species
abstract hydrogen atoms from the ribose backbone and/or
undergo β-elimination (<2 ꢀ 103 s-1).32 Hence, any direct
strand breaks were expected to result from peroxyl radicals. In
contrast to cleavage under anaerobic conditions, strand scission
from 5,6-dihydrouridin-6-yl radical (1) was lower in double-
stranded RNA (7) than the single-stranded substrate (9,
Table 2). With the exception of intranucleotidyl cleavage in
single-stranded RNA (9), the observed strand scission was very
low when 2 was photolyzed under aerobic conditions. Conse-
quently, we utilized additional oligonucleotide probes in order to
Direct Strand Scission under Anaerobic Conditions from
5,6-Dihydrouridin-6-yl (1) Flanked by Adenosines. Nucleo-
base radical reactivity in single (10) and double stranded (8)
substrates when flanked by adenosine was examined under
anaerobic conditions in order to probe the generality of the
chemistry observed when 1 is flanked by uridine (Table 1).
Adenosine was chosen over cytidine in order to provide a larger
structural distinction from uridine. Guanosine was not employed
due to possible complications arising from interactions between
the nucleobase and the photoexcited ketone.31 The qualitative
product distribution was very similar when 1 was produced in
duplex RNA in which it was flanked by A (8) as previously
observed forU (7). Directstrandscission resultingfrom oxidation
of the 50-adjacent nucleotide and the position at which the original
radical was generated yielded 30-fragments containing phosphate
groups at the 50-termini. 50-Fragments containing 30-phosphates
formed at both nucleotide positions. In addition, a slower moving
product consistent with a sugar fragment formed in conjunction
with oxidation of the 50-adjacent nucleotide was observed.
Although this product was not detected by mass spectrometry,
denaturing gel electrophoresis revealed that it reacted with
NaBH4 which is consistent with its assignment as 11b.30
Quantitative analysis of strand scission reinforces the simila-
rities between duplexes in which A (Table 1) or U flanks the 5,6-
dihydrouridin-6-yl radical (1).22 The percentage of strand scis-
sion that results from oxidation of the 50-adjacent nucleotide is
within experimental error when 1 is flanked by A or U (86.8 (
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Table 2. Direct Strand Scission under Aerobic Conditions
from 5,6-Dihydrouridin-6-yl (1) Flanked by Uridine
Scheme 2
absolute Yield (%)
rel. to
substratea
inter.
intra.
total
% inter.
anaerobic
ds (7)
2.2 ( 1.0 3.0 ( 1.1
1.3 ( 0.3 6.2 ( 1.0
5.2 ( 2.2 0.7 ( 0.5
1.4 ( 0.6 0.9 ( 1.1
1.1 ( 0.3 2.0 ( 0.7
5.2 ( 0.9 43.2 ( 20.0
7.5 ( 1.2 17.3 ( 1.5
5.9 ( 1.7 85.3 ( 15.4
2.3 ( 1.6 69.6 ( 21.6
3.1 ( 0.5 36.2 ( 11.6
0.1
0.4
0.2
0.2
0.2
1.4
ss (9)
ds (16)
ss (17)
ds (18)
ss (19)
Scheme 3
1.1 ( 0.8 9.2 ( 1.1 10.3 ( 1.7 10.1 ( 6.1
a The substrate is designated by its secondary structure (ss, ds).
substantiate that cleavage was not an artifact due to sample
handling. Substituting 5,6-dihydro-20-deoxyuridin-6-yl radical
(4) generated from the previously reported ketone (20, eq 2)
for the ribose analogue virtually eliminated intranucleotidyl
strand scission in single- (17) and double-stranded (16)
RNA.26,33 This is consistent with previous observations regard-
ing the reactivity of 4 in single- and double-stranded
oligodeoxynucleotides.12,26 Intranucleotidyl strand scission was
restored in single-stranded substrate (19) upon generation of 1
flanked by 20-deoxyuridine. These observations suggest that
intranucleotidyl hydrogen atom abstraction by the peroxyl
radical of 1 (21) in single-stranded substrates occurs and result
in strand breaks. Blocking intranucleotidyl strand scission in
single-stranded RNA does not result in an increase in cleavage at
the 50-adjacent nucleotide. However, it is possible that deactivat-
ing the intranucleotidyl pathway may increase hydrogen atom
abstraction from the 50-adjacent nucleotide but via a position (e.
g., C10) that does not lead to direct strand scission.12,34,35
For instance, we were only able to detect the phosphate end
group products resulting from intranucleotidyl cleavage by mass
spectrometry.30 Enzymic end group analysis confirmed that the
50-termini from single- and double-stranded substrates are ex-
clusively phosphate groups.30 Kinase analyses revealed that the
30-termini of the 50-fragments are composed mostly of phos-
phates, although there is a small amount of a product containing
an unidentified ribose fragment.30
Mechanism of 30-End Group Product Formation. The
previous report on the reactivity of 1 provided substantial
evidence that direct strand scission at the 50-adjacent uridine
resulted from C20-hydrogen atom abstraction.22 β-Fragmenta-
tion of phosphate from the resulting C20-radical to produce a
strand break was consistent with a large body of work initially by
von Sonntag and subsequently corroborated by other studies in
oligonucleotides and model compounds.2,5,28,36-38 Observation
of a 30-fragment containing a 50-phosphate was expected for this
mechanism. However, additional chemistry was required to
produce the 30-termini (30-phosphate (12) and 30-deoxy-20-
ketouridine (11a)) observed on the 50-fragment. We considered
the possibility that the 30-phosphate terminus resulted from C40-
deprotonation of 6, followed by β-fragmentation of the 50-
phosphate (22, Scheme 2). Deprotonation of olefin cation
radicals to produce allylic radicals can be very rapid.39
A
previously reported model of chorismate synthase in which an
allylic radical undergoes phosphate fragmentation provides addi-
tional precedent for producing RNA fragments containing 30-
phosphate termini.40 The requisite subsequent fragmentation
step is similar to the reaction that led directly to strand scission
and cleavage observed from C40-radicals in DNA.36
Formation of 11a within 7 (and related molecules in different
sequences) competes with 30-phosphate formation. One electron
reduction of the olefin cation radical (Scheme 3) would directly
yield the enol tautomer (23) of this product. Upon the basis of
known oxidation potentials of enol ethers and dG, electron
transfer from the purine should be thermodynamically downhill,
with one caveat.41-43 Hydrogen bonding to 6 could stabilize it
and reduce the driving force for electron transfer from
guanosine.44 There is ample precedence for hole migration in
DNA and this process was recently characterized in duplex RNA
as well.7,45-48 We investigated this possibility by measuring the
The low strand scission yields when 1 was produced under
aerobic conditions placed additional limits on product analysis.
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Table 3. Effect of Proximal Guanosines on the Ratio of 30-
Termini Products Resulting from Strand Cleavage by 5,6-
Dihydrouridin-6-yl (1)
Scheme 4
Figure 2. Effect of pH on (A) strand scission yield and (B) 30-end group
ratio from 5,6-dihydrouridin-6-yl (1) generated from 50-32P-7.
scission products resulting from internucleotidyl hydrogen atom
abstraction decreased less than 7% over the pH range between
7.2 and 3.8 (Figure 2A). However, the ratio of 30-phosphate
containing product to 11a varied significantly, with the largest
change occurring between pH 6.2 and 5.2 (Figure 2B). When the
pH is reduced to 3.8, the relative ordering of products is reversed,
and formation of the 30-ketone containing cleavage product
(11a) becomes favored over 30-phosphate (12). Reducing the
pH further to 1.9 resulted in a drastic reduction in the overall
yield of strand scission products.
These data are consistent with competitive deprotonation and
based upon the response of the product ratio to pH suggest that
C40-deprotonation in 6 is faster than those of the C10- and
hydroxyl group protons at higher pH. However, there is a reversal
in the relative ordering of rate constants at lower pH. It is difficult
to determine whether this is what one should expect because pKa
values of structurally similar cation radicals in H2O are not
available. Moreover, relevant kinetic constants are also scarce.
Although it is well-known that cation radicals are far more acidic
than their neutral parent molecules, the limited data available is
mostly for aromatic systems and pKa’s are often measured in
CH3CN. For instance, the pKa’s of a series of cation radicals
derived from stabilized enols range from 1.3 to 6.2 in
CH3CN.49,50 In contrast, the allylic proton in a lipid cation
radical was calculated to have a pKa ∼ 0 in the same solvent.51
Although these data do not address the deprotonation kinetics,
they are consistent with competing deprotonations from 6 to
form the final 30-termini products.
ratio of 30-end groups in a series of duplexes containing varying
amounts of guanosine in the vicinity of the nucleotide where the
radical cation forms (Table 3). More facile electron transfer
should result in a smaller ratio of 30-phosphate to ketone (11)
end products compared to the original duplex (7). The ease of
electron transfer was expected to correlate with the density of
guanosines proximal to the cation radical that would form at U9
following fragmentation (24 < 25 < 7 < 26). The overall strand
scission yields in the duplexes were within experimental error of
one another, suggesting that the photochemical generation of
5,6-dihydrouridin-6-yl (1) and subsequent strand scission are
unaffected by the changes in sequence. Although the ratio of the
products varied as a function of sequence, there was no clear
trend with respect to the anticipated driving force for electron
transfer.
Consequently, we turned our attention to an alternative
mechanism to explain formation of 11 (Scheme 4). We con-
sidered the possibility that deprotonation of the C10-carbon or
the hydroxyl group to yield R-keto radicals (27, 28, Scheme 4)
from 6 compete with proton loss from C40 to yield the product
containing a 30-phosphate terminus (e.g., 12, see Scheme 2).
Subsequent reduction and protonation of the R-keto radicals
would yield 11. Although we are unsure what the reducing agent
would be in this system, relative deprotonation rate constants
from C10, C40, and the 20-hydroxyl group could be affected by
pH. Changes in the relative rate constants of these product-
determining steps would be reflected in the ratio of 30-end group
products as the pH is varied. The combined yield of the strand
Hydrogen Atom Abstraction Is the Rate-Determining
Step in Direct Strand Scission. The rate constant for strand
cleavage (kCleave) from 5,6-dihydrouridin-6-yl (1) was estimated
in single-stranded (9) and double-stranded (7) RNA by
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measuring the effect of BME on the cleavage yield. The rate
constant was extracted from linear plots of the ratio of trapped
product to cleavage product versus BME concentration.30 The
trapping product was defined as the difference between the
amount of intact RNA in the presence of BME and the absence of
thiol. The slopes of these plots were the ratio of the rate constant
for BME trapping (kTrap = 2.6 ( 0.5 ꢀ 106 M-1 s-1) kCleave
determined in a previous study.25 Cleavage from 1 in double-
stranded RNA (7) was faster (kCleave = 31 ( 10 s-1) than in
single-stranded (9, kCleave = 13 ( 4 s-1) substrate.22,30 Assuming
that strand scission from 1 involves sequential hydrogen atom
abstraction and cleavage (Scheme 1), evaluation of the data in the
context of previously reported fragmentation rate constants
provides insight into the rate determining step for this process.
Radical fragmentation to yield a strand break is faster in single-
stranded than in double-stranded RNA.32 Oxidized abasic site
cleavage is also faster in single-stranded than in double-stranded
DNA.52,53 Faster cleavage in single-stranded substrates may be a
result of the complementary strand in a duplex acting as a
template for the reverse reaction, resulting in a slower observed
rate for strand scission. The opposite trend is observed in our
system. We interpret slower strand scission in single-stranded
RNA (9) as an indication that hydrogen atom abstraction by 1 is
the rate-determining step. Hydrogen atom abstraction by 1 could
be faster in the duplex (7) where the reactive partner is held in a
more rigid conformation.
conditions in the presence of 480 μM BME. These latter
products exhibited the expected shifts in m/z when 18O2 was
used, indicating that both products result from dioxygen trapping
of 1. Tandem lesion 35 was observed when 31 was photolyzed
under aerobic conditions in the absence of BME. MS/MS
analysis alternately in the presence of O2 or 18O2 revealed that
35 resulted from peroxyl radical (21) addition to the 50-adjacent
methyl uracil ring.30 This is consistent with what was observed
from the reactivity of the analogous DNA radical (4) under
aerobic conditions.12 Finally, the experiments were unaffected by
the stereochemistry of 2 in the dinucleotide, consistent with the
formation of products from a common intermediate, 5,6-dihy-
drouridin-6-yl (1).
Formation of Products from 5,6-Dihydrouridin-6-yl (1)
That Do Not Involve Strand Scission. Nucleobase damage
and abasic sites that do not result in direct strand scission are
detected in DNA and discerned from one another via alkaline
treatments of varying degrees of harshness.26 The inherent
lability of RNA to alkaline conditions is incompatible with this
approach, thus, hindering its product analysis. However, RNA
abasic sites (29) can be detected via the formation of strand
breaks upon treatment with aniline.34 Various 5-32P-labeled RNA
substrates containing 2 were photolyzed under aerobic or
anaerobic conditions. The remaining uncleaved oligonucleotide
was separated by denaturing polyacrylamide gel electrophoresis,
recovered, desalted, and treated with aniline. We analyzed single-
stranded and double-stranded RNA molecules in which 2 was
flanked by U or A. Abasic sites were not detected in any of these
substrates following generation of 1.30 The method was validated
by subjecting 50-32P-30, which was prepared using a variation of
Leumann’s method, to aniline cleavage treatment.30,34
’ CONCLUSIONS
As the major family of reactive species formed when OH
3
reacts with pyrimidines in RNA, a significant level of effort has
been spent studying nucleobase radical reactivity.1,54-58
A
number of pathways involving transfer of spin from a nucleobase
radical to a proximal (or distal) ribose ring were proposed. By
independently generating a nucleobase radical at defined posi-
tions in synthetic RNA, we unambiguously established that 5,6-
dihydrouridin-6-yl radical (1) produces direct strand breaks in an
intranucleotidyl manner and at the 50-adjacent phosphate. Direct
strand scission is more efficient under anaerobic conditions
because O2 trapping of the radical(s) is much faster than
hydrogen atom abstraction and subsequent fragmentation. A
potentially important outcome of this research is the preference
for strand scission in double-stranded RNA under anaerobic
conditions. This preference over single-stranded RNA is attri-
butable to faster hydrogen atom abstraction by 1 in the duplex
and is consistent with competitive kinetic studies. Because
nucleobase radicals such as 1 are the major species produced
Products ascribable to 1 containing modified nucleobases
were searched for via ESI-MS following photolysis of dinucleo-
tide 31.30 Methyl uridine was used as the 50-adjacent nucleotide
in order to potentially remove any ambiguity in product assign-
ment. The expected product (32) that contained 5,6-dihydrour-
idine was observed following photolysis of 31 (75 μM) under
anaerobic conditions in the presence of β-mercaptoethanol
(BME, 1 mM). Similarly, a mixture of the hydroxylated product
(33) and the respective hydroperoxide (34) was cleanly pro-
duced when the dinucleotide was photolyzed under aerobic
by OH , preferential cleavage by 5,6-dihydrouridin-6-yl radical
3
(1) in double-stranded RNA may be useful for distinguishing
between single- and double-stranded RNA in hydroxyl radical
cleavage experiments. If the preference for direct strand scission
under anaerobic conditions in duplex RNA by 1 is general for
nucleobase radicals, single- and double-stranded RNA will be
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distinguished from one another by comparing relative cleavage
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3
aerobic and anaerobic conditions. Double-stranded regions will
be discernible by relative increases in strand scission under
anaerobic conditions. Selective 20-hydroxyl acylation analyzed
by primer extension (SHAPE) is currently the most general
chemical method available for determining secondary RNA
structure.59,60
Enhanced cleavage under anaerobic conditions by 1 is also
contrary to the generally accepted oxygen enhancement by
ionizing radiation, which is often associated with the ability of
O2 to “fix” (prevent repair by thiols) DNA radicals.1 Overall
strand scission in poly(U) induced by ionizing radiation in-
creases under aerobic conditions. This suggests that nucleobase
radicals, despite their relatively high yield, are not major con-
tributors to direct strand scission under aerobic conditions.
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’ ASSOCIATED CONTENT
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S
Supporting Information. Experimental procedures,
b
mass spectral data, and sample autoradiograms. This material is
available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
mgreenberg@jhu.edu
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Author Contributions
†These authors contributed equally.
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