Direct strand scission from a nucleobase radical in RNA

Article abstract of DOI:10.1021/ja100281x

"Chemical equation presented" RNA oxidation is important in the etiology of disease and as a tool for studying the structure and folding kinetics of this biopolymer. Nucleobase radicals are the major family of reactive intermediates produced in RNA exposed to diffusible species such as hydroxyl radical. The nucleobase radicals are believed to produce direct strand breaks by abstracting hydrogen atoms from their own and neighboring ribose rings. By independently generating the formal C5 hydrogen atom addition product of uridine in RNA, we provide the first chemical characterization of the pathway for direct strand scission from an RNA nucleobase radical. The process is more efficient under anaerobic conditions. The preference for strand scission in double-stranded RNA over single-stranded RNA suggests that this chemistry may be useful for analyzing the secondary structure of RNA in hydroxyl radical cleavage experiments if they are carried out under anaerobic conditions.

Full text of DOI:10.1021/ja100281x

Published on Web 02/25/2010
Direct Strand Scission from a Nucleobase 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
Received January 12, 2010; E-mail: mgreenberg@jhu.edu
Historically, homopolymers of RNA were used as model
substrates in studies of nucleic acid damage by γ-radiolysis.1 More
recently, RNA oxidation has become important in its own right
because of its association with disease and its use as a tool for
probing RNA folding and structure.2–6 Hydroxyl radical (HO ·) is
a primary damaging agent, and direct strand scission is proposed
to result from as many as 40% of its reactions with RNA. A variety
of experiments have established that π-bond addition is the major
reaction pathway for HO· (and H·) with RNA, and the resulting
nucleobase radicals account for as much as 90% of the reactions.1,7
Consideration of strand break efficiency and hydroxyl radical
reactivity suggests that nucleobase radicals and/or their respective
peroxyl radicals abstract hydrogen atoms from ribose rings because
direct strand scission requires formation of sugar radicals.1,8
Characterization of these pathways using HO · is complicated by
the unselective nature by which diffusible reactive species react
with nucleic acids. Our understanding of DNA oxidation has been
greatly improved by independently generating reactive intermediates
within synthetic oligonucleotides.9,10 We now report the reactivity
of the first independently synthesized nucleobase RNA radical and
its role in direct strand scission.
photocleavage of monomeric 2 was independent of O2, suggesting
that the observed trend is indicative of the effect of O2 on one or
more steps in the mechanism for strand scission from 1.11
Table 1. Direct Strand Scission under Anaerobic Conditionsa
absolute yield (%)
substrate
Inter
Intra
total
% Interb
3
4
5
6
7
3.3 ( 0.7
38.9 ( 3.0
3.6 ( 1.6
25.6 ( 3.5
4.4 ( 0.5
17.3 ( 7.2
6.0 ( 2.2
13.2 ( 3.6
4.0 ( 2.0
10.9 ( 1.6
20.7 ( 7.2
45.0 ( 4.8
16.8 ( 5.2
29.6 ( 1.5
15.3 ( 1.6
17.9 ( 8.4
86.8 ( 3.7
20.7 ( 4.2
86.3 ( 7.1
29.0 ( 4.0
a 5′-32P substrates were photolyzed for 4 h. Each value is the average
of at least three experiments. Each experiment consisted of three
replicates. b Percentage of total direct strand scission produced at the
“Inter” position.
Under anaerobic conditions, direct strand scission products corre-
sponding to oxidation at the nucleotide at which 1 (or 9) was generated
(“Intra”) and at the 5′-adjacent uridine (“Inter”) in 5′-32P-3 and 5′-
32P-4 were observed, although scission was more efficient overall in
the duplex 4 (Table 1, Figure 1). The ratio of strand scission at the
sites depended upon whether 1 was produced in single- or double-
stranded RNA, although it was independent of photolysis time for a
Scheme 1
Hydroxyl radical and hydrogen atom preferentially add to the
C5 position of pyrimidines (Scheme 1). We independently generated
the monomeric form of the C5 hydrogen atom adduct of uridine,
5,6-dihydrouridin-6-yl radical (1), via Norrish type-I photocleavage
of nucleoside 2.11 Oligonucleotides containing 2 were prepared via
automated solid-phase oligonucleotide synthesis.12 Direct strand
break formation following photolysis of 2 (350 nm, 4 h) was
examined in single- and double-stranded RNA under aerobic and
anaerobic conditions. The amount of direct strand scission under
aerobic conditions was less than one-seventh of that observed when
1 was produced in a duplex (4) in the absence of O2. The
Figure 1. Denaturing PAGE analysis of direct strand scission upon photolysis
of (A) 5′-32P-3, (B) 5′-32P-4, and (C) 5′-32P-7 under anaerobic conditions.
particular substrate. Strand scission occurred at the 5′-adjacent uridine
>85% of the time when 5,6-dihydrouridin-6-yl radical (1) was produced
in double-stranded RNA (4), but strand scission at the site where 1
was generated accounted for >80% of strand breaks in the single-
stranded substrate (3). Products containing 3′- or 5′-phosphate termini
were identified via enzymatic dephosphorylation and comparison with
independently synthesized markers. The 5′-termini consisted solely of
phosphate groups. However, the 3′-termini contained nucleotide
fragments in addition to phosphate end groups. The structure of these
other 3′-end groups have not yet been determined.
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3668 J. AM. CHEM. SOC. 2010, 132, 3668–3669
10.1021/ja100281x  2010 American Chemical Society

C O M M U N I C A T I O N S
Qualitatively, the level of direct strand scission from 1 is significantly
higher than from the analogous radical 5,6-dihydro-2′-deoxyuridin-6-
yl (9) in oligodeoxynucleotides.13,14 We speculated that the presence
of the 2′-hydroxyl group, which results in a significant reduction in
the C2′-H bond dissociation energy, played a vital role in facilitating
transfer of the radical from the nucleobase to the sugar.15 The
importance of the 2′-hydroxyl group on direct strand scission was
verified by replacing the 5′- and 3′-adjacent uridine nucleotides with
2′-deoxyuridine. Photolysis of 5′-32P-5 resulted in a more than 10-
fold reduction in strand scission at the 5′-adjacent nucleotide and a
2-fold increase at the position where 1 was generated (Table 1). In
contrast, the amount of direct strand scission at the 5′-adjacent
nucleotide when 9 was flanked by uridine (5′-32P-6) was restored to a
level comparable to that observed in 4.
The kinetics of the strand scission process was probed using
ꢀ-mercaptoethanol (BME) as a competitor. Direct strand scission in
5′-32P-4 was quenched by micromolar concentrations of the thiol. The
thiol can prevent strand cleavage by trapping 1 and/or the subsequently
formed ribose radical(s) (e.g., 10). The amount of RNA radical trapped
by the thiol was defined as the difference in the amount of strand
scission with and without BME. Plotting the ratio of thiol-trapped
radical to strand scission as a function of BME concentration yielded
a straight line whose slope represents the ratio of rate constants for
thiol trapping and the rate-determining step in strand scission.12
Depending upon whether the estimated rate constant for BME trapping
of monomeric 1 (2.6 × 106 M-1 s-1)11 or a generic rate constant for
thiol reaction with an alkyl radical (8 × 106 M-1 s-1)18 is used, these
data indicate that the rate constant of the rate-limiting step in strand
scission is between 29 and 90 s-1. Further investigation is required in
order to determine whether hydrogen atom abstraction by 1 or
phosphate elimination is the rate-determining step in strand scission.
In summary, we have characterized the mechanism by which an
RNA nucleobase radical is transformed into a direct strand break
under anaerobic conditions. The major pathway in duplex RNA
involves internucleotidyl hydrogen atom abstraction from the C2′
position of the 5′-adjacent nucleotide. A thiol competition experi-
ment indicated that direct strand scission from 1 is too slow to
compete with the millimolar concentration of thiol present in cells.
However, the more efficient direct strand break formation in duplex
RNA in comparison with single-stranded substrate under anaerobic
conditions may prove useful for extracting additional structural
information from hydroxyl radical cleavage experiments.
Scheme 2
These observations and examination of molecular models
(Spartan ’02) led us to propose that the major pathway for direct
strand scission from 5,6-dihydrouridin-6-yl radical (1) in duplex
RNA under anaerobic conditions involves C2′ hydrogen atom
abstraction from the 5′-adjacent uridine (Scheme 2). This hypothesis
was supported by photolysis of 5′-32P-7, in which the 5′-adjacent
uridine was deuterated at C2′. Formation of 1 in 5′-32P-7 resulted
in more than an 8-fold reduction in the absolute amount of direct
strand scission at the 5′-adjacent C2′-deuterated uridine relative to
what was produced in 5′-32P-4 (Table 1). Although the level of
strand scission at the nucleotide where 1 was generated increased
in the deuterated substrate, the increase did not fully compensate
for the diminution in strand scission at the 5′-adjacent nucleotide.
Hence, we cannot rule out an increased contribution to the radical’s
overall reactivity at a position within 1 or an adjacent nucleotide
that does not result in direct strand scission (e.g., C1′) when the
C2′ hydrogen of the 5′-adjacent uridine is deuterated.
Acknowledgment. We are grateful for generous support from
the National Institute of General Medical Sciences (GM-054996).
M.J.E.R. thanks the NIGMS for a Research Supplement To Promote
Diversity in Health-Related Research.
Supporting Information Available: Procedures for the synthesis
of all compounds and other experiments; ESI-MS of modified oligo-
nucleotides; and sample phosphorimages of enzymatic end group
analysis, deuterium effect on strand scission, and thiol trapping. This
material is available free of charge via the Internet at http://pubs.acs.org.
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