Photochemical generation of ribose abasic sites in RNA oligonucleotides

Article abstract of DOI:10.1021/ol050120h

(Chemical Equation Presented) A general method for the photochemical generation of ribose abasic sites within RNA oligonucleotides is reported. Photochemically caged nucleoside phosphoramidite analogues were prepared and incorporated into RNA oligonucleot

Full text of DOI:10.1021/ol050120h

ORGANIC
LETTERS
2005
Vol. 7, No. 8
1493-1496
Photochemical Generation of Ribose
Abasic Sites in RNA Oligonucleotides
John D. Trzupek and Terry L. Sheppard*
Department of Chemistry and The Robert H. Lurie ComprehensiVe Cancer Center,
Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208-3113
t-sheppard@northwestern.edu
Received January 19, 2005
ABSTRACT
A general method for the photochemical generation of ribose abasic sites within RNA oligonucleotides is reported. Photochemically caged
nucleoside phosphoramidite analogues were prepared and incorporated into RNA oligonucleotides by automated RNA synthesis. Irradiation
of the modified RNA at 350 nm efficiently produced ribose abasic sites at specific sites within RNA sequences. The current approach offers
a chemical route to RNA abasic lesions for RNA biochemical studies.
The discovery of the Tetrahymena self-splicing intron1 and
the catalytic RNA of RNase P2 highlighted RNA’s dual role
as an informational and catalytic macromolecule. Since then,
RNA structural biology has shown that RNA adopts complex
folded structures, reminiscent of proteins. In particular, the
solution of the three-dimensional structure of the ribosome3
has demonstrated that RNA molecules play a catalytic role
in the chemistry of peptide bond formation in all cells.4
nucleotide chemical structure is essential for maintaining the
integrity of the genome and gene expression. For this reason,
DNA damage and repair mechanisms have received consid-
erable recent attention.6 For example, DNA “abasic sites”,
which result from hydrolysis of nucleotide N-glycosidic
bonds or oxidation of sugar moieties in DNA nucleotides,7
are premutagenic lesions that stall DNA and RNA poly-
merases.8 DNA repair enzymes have evolved to excise abasic
lesions and facilitate insertion of the correct nucleotide at
the damage site.
RNA damage and repair mechanisms, in contrast, have
received considerably less attention. Ribose abasic sites (Z,
Scheme 1), resulting from N-glycosidic bond cleavage in
RNA, may interfere with RNA coding and folding. RNA
abasic sites are generated in cellular RNA by a class of
Pairing and stacking of nucleotide bases plays an important
role in defining the three-dimensional structure, and resulting
function, of DNA and RNA.5 Thus, the preservation of
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10.1021/ol050120h CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/23/2005

Scheme 1. Photochemical Generation of Ribose Abasic Sites
Scheme 2. Synthesis of Phosphoramidites 1a and 1b
N-glycosylase proteins called ribosomal inactivating proteins
(RIPs).9 A prototypic example is the toxin ricin.10 The ricin
A-chain (RA) targets ribosome inactivation by specific
depurination of A4324 in the 28S ribosomal RNA (rRNA).
Structural studies of the sarcin/ricin (S/R) loop11 demon-
strated that A4324 plays a critical role in molecular recognition
within the ribosome. Thus, the generation of a ribose abasic
site by ricin blocks ribosomal translocation and leads to cell
death.12 The importance of A4324 was further underscored
by a recent report of an RNA repair mechanism for the S/R
rRNA domain.13
To facilitate biochemical studies of the ribosome and RIP
action, we developed a chemical method for the generation
of ribose abasic sites within RNA. A nonnatural tetrahydro-
furan mimic of ribose abasic sites has been prepared.14
Although this analogue serves as an effective model system,15
it does not contain the C-1′ hemiacetal functionality observed
in natural ribose abasic sites. A current approach to ribose
abasic sites involves treatment of target RNAs with an RIP,
which is limited by the specificity and availability of the
enzyme.16 On the basis of methods for the site-specific
generation of DNA aldehyde17 and oxidized abasic sites,18
we now report a photochemical method for the introduction
of unique ribose abasic sites within RNA oligonucleotides.
We describe the synthesis of two C-1 “caged” analogues (1a
and 1b, Scheme 2), their incorporation into RNA oligo-
nucleotides by solid-phase RNA synthesis, and efficient
photolytic deprotection to produce ribose abasic sites (Scheme
1).
Two target phosphoramidites (1a and 1b, Scheme 2) were
selected as precursors of ribose abasic sites. Each analogue
contained a C-1 benzyl ether to enable selective photochemi-
cal release of the desired abasic site. The synthesis of the
nitrobenzyl phosphoramidite (1a, Scheme 2) commenced
with SnCl4-promoted glycosidation of 8 to yield the ano-
merically pure, protected ribofuranoside 9a in 89% yield.
The ribofuranoside 10a was prepared by ammonolysis of
the benzoyl protecting groups. Regioselective tritylation of
10a was accomplished by slow addition of DMTCl, affording
the desired ether 11a in good yield. Silylation conditions,
based on reported methods,19 yielded a mixture of the 2′-
O-silyl nucleoside (12a) and the isomeric 3′-O-silyl analogue
(13a). The desired 2′-O-isomer (12a) was isolated by
chromatography and converted to 1a by standard RNA
phosphitylation conditions.20 The 3′-O-silyl isomer (13a) was
utilized for the preparation of a solid-supported analogue for
the synthesis of 3′-terminal caged RNA.21 Analogue 1b,
which was based on the nitroveratryl protection group, was
prepared by a parallel synthetic route (Scheme 2).
(9) (a) Girbes, T.; Ferreras, J. M.; Arias, F. J.; Stirpe, F. Mini-ReV. Med.
Chem. 2004, 4, 461-476. (b) Barbieri, L.; Battelli, M. G.; Stirpe, F. Biochim.
Biophys. Acta 1993, 1154, 237-282.
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Biochem. J. 1975, 146, 127-131.
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Endo, Y. EMBO J. 1999, 18, 6522-6531.
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1994, 4, 1715-1720.
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Acad. Sci. U.S.A. 1996, 93, 11522-11527.
(18) (a) Kotera, M.; Bourdat, A.-G.; Defrancq, E.; Lhomme, J. J. Am.
Chem. Soc. 1998, 120, 11810-11811. (b) Tronche, C.; Goodman, B. K.;
Greenberg, M. M. Chem. Biol. 1998, 5, 263-271. (c) Lenox, H. J.; McCoy,
C. P.; Sheppard, T. L. Org. Lett. 2001, 3, 2415-2418.
(19) Allerson, C. R.; Chen, S. L.; Verdine, G. L. J. Am. Chem. Soc.
1997, 119, 7423-7433.
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4922.
(17) (a) Groebke, K.; Leumann, C. HelV. Chim. Acta 1990, 73, 608-
617. (b) Peoc’h, D.; Meyer, A.; Imbach, J. L.; Rayner, B. Tetrahedron Lett.
1991, 32, 207-210. (c) Shishkina, I. G.; Johnson, F. Chem. Res. Toxicol.
2000, 13, 907-912.
(20) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res. 1990,
18, 5433-5441.
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Org. Lett., Vol. 7, No. 8, 2005

Figure 1. Photochemical generation of ribose abasic sites. 5′-
Radiolabeled oligonucleotide (2 or 3) was irradiated at 350 nm in
phosphate-buffered saline (PBS) buffer and analyzed by 20%
dPAGE. (A) Nitrobenzyl-caged RNA (2) remaining was 93%, 70%,
49%, 36%, 23%, 10%, and 7% for lanes 1-7, respectively. Lane
8: reaction with biocytin hydrazide (5 mM final concn, 95 °C, 30
min, 57% conversion). (B) Nitroveratryl-caged RNA (3) remaining
was 89%, 75%, 61%, 56%, 53%, 25%, 14%, and 10% for lanes
1-8, respectively.
Several model RNA sequences were designed to demon-
strate the use of analogues 1a and 1b for the synthesis of
ribose abasic sites. Sequences 2 and 4 contained the
nitrobenzyl-caged abasic site at position X (from 1a, Scheme
1). Oligonucleotide 2 was designed as a mimic of the S/R
domain in 28S rRNA, and oligonucleotide 4 served as a
longer sequence to validate the method. Sequences 3 and 5
were analogous to 2 and 4, respectively, but were prepared
with the nitroveratryl analogue, 1b (Y, Scheme 1).
Phosphoramidite analogues 1a and 1b were used to
introduce the caged analogues into RNA oligonucleotides
(2 and 4 or 3 and 5, respectively) by standard RNA solid-
phase synthesis methods.22 Analogue 1a was incorporated
in modest yield (53%), even with enhanced coupling times.
However, 1b was introduced with high coupling yields
(98%). RNA nucleotide coupling yields were ∼98% after
introduction of each caged analogue.
Caged oligonucleotides 2-5 were deprotected using
standard conditions: RNA was treated with 28% NH4OH/
EtOH for 24 h followed by immediate desilylation. Standard
TBAF desilylation conditions20 led to modified RNA prod-
ucts. However, deprotection with triethylamine trihydro-
fluoride (TEA‚3HF, ∼24 h at 65 °C) provided RNA products
cleanly. Deprotected oligonucleotides were purified by
denaturing polyacrylamide gel electrophoresis (20% dPAGE;
29:1 acrylamide/bisacrylamide, 8 M urea), followed by
reverse-phase HPLC (RP-HPLC). A single oligonucleotide
product was observed after purification. RNA oligonucleo-
tides 2-5 were characterized by mass spectrometry (MALDI-
TOF MS). All caged oligonucleotides showed the expected
molecular masses (see Supporting Information).
Figure 2. RP-HPLC and MALDI-TOF MS characterization of
photochemical deprotection for (A) oligonucleotide 2 and (B)
oligonucleotide 3. (C) Products from photolytic cleavage of each
caged species (2 and 3) were combined and coinjected. Retention
times of the corresponding oligonucleotides were 5.7, 5.3, and 4.4
min for 2, 3, and 6, respectively.
in Scheme 1). Oligonucleotides 2 and 3 were 5′-radiolabeled
and independently irradiated at 350 nm. Reaction aliquots
were removed at specific times and analyzed by dPAGE.
As shown in Figure 1, caged RNAs 2 (Figure 1A) and 3
(Figure 1B) underwent a time-dependent gel mobility shift
upon UV irradiation. Deprotection half-lives of 2.3 min (2)
and 2.2 min (3) were determined by quantification of the
dPAGE analysis was used to evaluate the efficiency of
photolytic deprotection of oligonucleotides 2 and 3 to
produce RNA containing a unique ribose abasic site (6, Z
Org. Lett., Vol. 7, No. 8, 2005
1495

gel image. To support a preliminary assignment of 6,
oligonucleotides 2 and 3 were irradiated and treated with
the aldehyde-reactive probe, biocytin hydrazide (BH). BH
was designed to trap aldehyde abasic sites by hydrazone
formation.23 Caged 2 or 3 remained unmodified by BH (<1%
hydrazone formation). However, decaging of oligonucleotide
2 or 3 led to a product that was efficiently labeled by BH
(lane 8, Figure 1A). Caged oligonucleotides 4 and 5 also
were subjected to photolytic treatment and gel electrophoretic
analysis. Due to the greater length of 4 and 5, the corre-
sponding product oligonucleotide (7, Scheme 1) comigrated
with the starting oligonucleotides during gel analysis.
However, 4 and 5 displayed a UV-induced, time-dependent
gel mobility shift when incubated with BH (Supporting
Information). The gel electrophoresis and chemoselective
labeling system provided validation of the approach (Scheme
1), but more rigorous characterization standards were applied.
Formation of ribose abasic sites in RNA oligonucleotides
was validated directly by RP-HPLC and MALDI-TOF MS
analysis. Caged oligonucleotides 2 and 3 exhibited RP-HPLC
retention times of 5.7 and 5.3 min, respectively (Figures 2A
and 2B). After UV irradiation (350 nm) for 25 min, each
oligonucleotide was quantitatively converted to a single peak
with a retention time of 4.4 min. Products from the decaging
reactions of 2 and 3 were isolated, mixed, and subjected to
RP-HPLC analysis. The products coeluted with a retention
time of 4.4 min (Figure 2C), indicating that they were
identical. In a separate experiment, oligonucleotides 2 and
3 were irradiated, and the resulting products were purified
and characterized by mass spectrometry. MALDI-TOF MS
analysis of the decaged products resulted in m/z values of
3119.6 for 2 and 3119.9 for 3, which were in excellent
agreement with the predicted mass for 6 (m/z ) 3119.9).
Oligonucleotides 4 and 5 were subjected to a parallel RP-
HPLC and MALDI-TOF MS analysis (Supporting Informa-
tion). RP-HPLC coinjection experiments demonstrated that,
upon irradiation, 4 and 5 produced the same product, which
had the mass expected for 7 (calcd 5229.1 m/z; found 5229.1
(4) and 5229.8 (5)).
Further analysis showed that ribose abasic sites in RNA,
like their DNA counterparts,15 readily underwent strand
scission by â-elimination. For example, when purified 6 was
concentrated to dryness, three peaks with retention times of
3.0, 3.3, and 4.4 min were observed by RP-HPLC. MALDI-
TOF MS data for the two major products were in good
agreement with the masses expected for â-elimination at
position Z. Thus, to avoid strand scission, oligonucleotides
containing ribose abasic sites should be handled at low
temperatures and under controlled conditions.
In summary, we have developed a general method for the
site-specific introduction of ribose abasic sites within RNA
oligonucleotides. Using a caged phosphoramidite reagent, a
stable nucleotide analogue was introduced into RNA by
solid-phase synthesis. The ribose abasic site lesion was
subsequently revealed by UV irradiation. The system permits
the insertion of ribose abasic sites at any site within an RNA
oligonucleotide, which will provide a new reagent for RIP
mechanistic studies. With appropriate modification, our
approach should be compatible with other high-yielding RNA
synthesis strategies.24 In addition, our method, combined with
standard RNA splint-ligation methods,25 should permit
studies of ribose abasic sites within large RNAs. Finally,
the C-1 aldehyde of the ribose abasic site may offer a unique
chemical handle for chemoselective modification reactions
within RNA sequences.26
Acknowledgment. This work was supported by the
Robert H. Lurie Comprehensive Cancer Center of North-
western University. The MALDI-TOF mass spectrometer
was purchased with funds provided by an NIH Scientific
Instrumentation grant (1-S10-RR13810).
Supporting Information Available: Detailed experi-
mental protocols and spectral data for all compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL050120H
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