Chemical synthesis of U1 snRNA derivatives

Article abstract of DOI:10.1021/ol401917r

U1 snRNA is an interesting biological tool for splicing correction and regulation of gene expression. However, U1 snRNA has never been chemically synthesized. In this study, the first chemical synthesis of U1snRNA and its analogues was carried out. Moreov

Full text of DOI:10.1021/ol401917r

ORGANIC
LETTERS
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Vol. XX, No. XX
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Chemical Synthesis of U1 snRNA
Derivatives
Akihiro Ohkubo,*,† Yasushi Kondo,‡ Makoto Suzuki,† Haruki Kobayashi,†
Takashi Kanamori,† Yoshiaki Masaki,† Kohji Seio,† Kiyoshi Nagai,‡ and MitsuoSekine*,†
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku,
Yokohama 226-8501, Japan, and MRC Laboratory of Molecular Biology, Francis Crick
Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, U.K.
aohkubo@bio.titech.ac.jp; msekine@bio.titech.ac.jp
Received July 8, 2013
ABSTRACT
U1 snRNA is an interesting biological tool for splicing correction and regulation of gene expression. However, U1 snRNA has never been chemically
synthesized. In this study, the first chemical synthesis of U1snRNA and its analogues was carried out. Moreover, it was found that the binding affinity
of the modified U1 snRNA with an ethylene glycol linkage to snurportin 1 (nuclear import adaptor) was as high as that of the unmodified RNA.
Uridine-rich small nuclear RNAs (U snRNAs) are
involved in very important pre-mRNA splicing events in
the nucleus of eukaryotic cells.1 Among them, U1snRNA can
recognize the 50-splice site of pre-mRNA. At the first splicing
step, U1snRNA directly binds to the splice site by hybridiza-
tion between the 50-terminal eight nucleotides of U1snRNA
and the complementary sequence of pre-mRNA.2
Splicing disorders are estimated to account for about
15% of disease-causing mutations in humans, and the
majority of genetic mutations are point mutations within
the sequences of splice sites.3 A subset of these defects
should result from mismatched base pairs between
U1snRNA and mutant pre-mRNAs. It was reported in a
recent study that mutant pre-mRNA splicing could be
promoted by modified U1snRNAs that were prepared
using the biological technique in the cell and could hybri-
dize with the mutant pre-mRNA.4 These results indicate
that modified U1snRNA could be useful for splicing
correction in gene therapy.
Additionally, a modified U1snRNA containing the
sequence complementary to the 30-terminal exon of the
targeted gene can downregulate the targeted gene expres-
sion via U1 interference (U1i).5 This downregulation
results from inhibition of mRNA polyadenylation by the
U1 snRNA-binding protein (U1ꢀ70K protein). However,
U1 snRNA has never been chemically synthesized because
U1 snRNA is very long (164 nt) and contains a compli-
cated 50-terminal 2,2,7-N-trimethylguanosine (m32,2,7G)
(5) (a) Abad, X.; Vera, M.; Jung, S. P.; Oswald, E.; Romero, I.; Amin,
V.; Fortes, P.; Gunderson, S. I. Nucleic Acids Res. 2008, 36, 2338–2352.
(b) Grunweller, A.; Hartmann, R. K. ChemBioChem 2009, 10, 1599–
1601. (c) Kanoepfel, A. S.; Abad, A.; Abad, X.; Fortes, P.; Berkhout, B.
† Tokyo Institute of Technology.
‡ MRC Laboratory of Molecular Biology.
€
(1) (a) Jurica, M. S.; Moore, M. J. Mol. Cell 2003, 12, 5–14. (b) Wahl,
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(2) Pomeranz Krummel, D. A.; Oubridge, C.; Leung, A. K. W.; Li, J.;
Antiviral Res. 2012, 94, 208–216.
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Chim. Acta 2001, 84, 3773–3795. (b) Shiba, Y.; Masuda, H.; Watanabe,
N.; Ego, T.; Takagaki, K.; Ishiyama, K.; Ohgi, T.; Yano, J. Nucleic Acids
Res. 2007, 35, 3287–3296. (c) Lang, K.; Micura, R. Nat. Protoc. 2008, 3,
1457–1466. (d) Huber, J.; Cronshagen, U.; Kadokura, M.; Marashally,
Nagai, K. Nature 2009, 458, 475–481.
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Bresolin, N. Hum. Genet. 2001, 109, 73–84.
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182. (b) Alanis, E. F.; Pinotti, M.; Mas, A. D.; Balestra, D.; Cavallari,
N.; Rogalska, M. E.; Bernardi, F.; Pagani, F. Hum. Mol. Genet. 2012, 21,
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r
10.1021/ol401917r
XXXX American Chemical Society

Figure 2. Chemical structure of (a) m32,2,7G cap, (b) pyropho-
sphate analogue, and (c) ethylene glycol analogue.
Figure 1. Structure of U1snRNA.
cap (Figures 1 and 2a).6 The m32,2,7G cap can bind to the
nuclear import adaptor snurportin 1 (SPN1) and plays an
important role in transporting UsnRNAs between the
cytoplasm and the nucleus. In this study, we carried out
the chemical synthesis of U1snRNA and modified
U1snRNAs containing the m32,2,7G cap analogues for
the first time, as shown in Figure 2.
Figure 3 depicts the strategy for synthesizing U1snRNA
with a fluorescent label at the 30-terminus. First, we
synthesized 50-short RNA containing the m32,2,7G cap by
chemical reactions on polymer supports (Schemes 1 and 2)
and 30-long RNA by biochemical methods using T7 RNA
polymerase and a chemical reaction in solution phase
(Scheme 3). We subsequently generated the complex
formed between 50-short RNA, 30-long RNA, and the
complementary splint DNA. After hybridization, the liga-
tion reactions were carried out using T4 DNA ligase.7
In the synthesis of 50-short RNAs, we used the highly
cross-linked polystyrene resin8 1 that has a silyl-type
linker,9 which can be cleaved under neutral conditions,
phosphoryl10 and phosphityl reagents 2ꢀ4 (Figure 4). The
synthesis of 50-short RNA I is shown in Scheme 1. After
chain elongation of the RNA sequence using 20-O-
TBDMS RNA phosphoramidite units on the resin by
using an RNA synthesizer, the treatment of resin 5 with
100 equiv of diphenyl phosphonate in pyridine was fol-
lowed by hydrolysis mediated by a H-phosphonate
Figure 3. Strategy for U1snRNA synthesis.
(7) Moore, M. J.; Sharp, P. A. Science 1992, 256, 992–997.
(8) McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32, 4069–
4072.
(9) (a) Ohkubo, A.; Seio, K.; Sekine, M. J. Am. Chem. Soc. 2004, 126,
10884–10896. (b) Ohkubo, A.; Kasuya, R.; Aoki, K.; Kobori, A.;
Taguchi, H.; Seio, K.; Sekine, M. Bioorg. Med. Chem. 2008, 16, 5345–
5351.
(10) (a) Ohkubo, A.; Aoki, K.; Seio, K.; Sekine, M. Tetrahedron Lett.
2004, 45, 979–982. (b) Ohkubo, A.; Tago, N.; Yokouchi, A.; Nishino, Y.;
Yamada, K.; Tsunoda, H.; Seio, K.; Sekine, M. Org. Lett. 2012, 14, 10–
13.
Figure 4. Structure of resin 1, phosphoryl and phosphityl re-
agents 2ꢀ4.
B
Org. Lett., Vol. XX, No. XX, XXXX

compound. The compound was subsequently oxidized
using (2R,8S)-(þ)-(camphorsulfonyl)oxaziridine in the
presence of N,O-bis(trimethylsilyl)acetamide (BSA). The
50-terminal pyrophosphate bond was formed by the reac-
tion of the protected oligonucleotide immobilized on the
resin with compound 2 for 15 min, as shown in Scheme 1.
After deprotection of the 2-cyanoethyl, sulfonylethyl, and
N-acyl groups, the 50-terminal capping reaction was per-
formed by treating residue 6 with m32,2,7G phosphorimi-
dazolide unit 7.11 The 20-TBDMS groups of the oligomer
were subsequently deprotected, and the oligomer was
released from the resin by treatment with 1 M tetrabuty-
lammonium fluoride (TBAF) in the presence of 0.5 M
AcOH for 24 h. Although the only 20-TBDMS groups of
pseudouridine residues could not be cleaved under the
deprotection conditions using TBAF, the TBDMS groups
were easily removed by treatment with a 20% acetic acid
aqueous solution for 3 h.12 The target 50-short RNA I was
isolated by anion-exchange HPLC in 2% yield and charac-
terized by MALDI-TOF mass spectrometry. Additionally,
the 50-short RNA II having a pyrophosphate group was
obtained as a byproduct in 3% isolated yield.
The yield of 50-short RNA III was much higher than that of
RNA with an unmodified m32,2,7G cap.
Scheme 2. Synthesis of 50-Short RNA III Containing an
m32,2,7G Cap Analoguea
a The oligonucleotide sequence is AmUmACΨΨAC.
Scheme 1. Synthesis of 50-Short RNA I Containing an m32,2,7G
Scheme 3. Synthesis of 30-Long RNA
Capa
The 30-long RNA was synthesized by RNA transcrip-
tion using T7 RNA polymerase, as shown in Scheme 3. In
this RNA transcription, we combined the desired RNA
sequence and the hammerhead ribozyme sequence because
efficient synthesis requires the RNA to have a purine-rich
sequence at the 50-terminus.13 The 50-sequence of hammer-
head ribozyme was efficiently eliminated under the tran-
scription conditions in the presence of Mg2þ. After PAGE
purification, we introduced a fluorescent label into the 30-
terminus of the RNA by treatment with NaIO4 and
fluorescein-5-thiosemicarbazide in 65% isolated yield.
For the final step, 50-phosphorylation was carried out
using T4 polynucleotide kinase.
a The sequence of oligonucleotide is AmUmACΨΨAC.
The 50-short RNAs IꢀIII were ligated with the 30-long
RNA by T4 DNA ligase to obtain the U1 snRNA I and its
analogues (U1snRNA 2 with a pyrophosphate linkage and
U1 snRNA III with an ethylene glycol linkage) with a
fluorescent label in the presence of the complementary
splint DNA. The target U1 snRNAs IꢀIII were isolated by
PAGE in 14, 28, and 30% yields, respectively, and char-
acterized by MALDI-TOF mass spectrometry. These
In the synthesis of 50-short RNA III containing an
m32,2,7G cap analogue, the resin 8 was prepared by chain
elongation using phosphityl reagents 3and 4on resin 1.Resin
8 was treated with DBU and MeNH2 to obtain resin 9. After
capping using phosphorimidazolide unit 7, the oligomer was
deprotected and released from the resin in 12% isolated yield.
(11) Ohkubo, A.; Sasaki, K.; Noma, Y.; Tsunoda, H.; Seio, K.;
Sekine, M. Bioorg. Med. Chem. 2009, 17, 4819–4824.
(12) Kawahara, S.; Wada, T.; Sekine, M. J. Am. Chem. Soc. 1996,
118, 9461–9468.
(13) Price, S. R.; Ito, N.; Oubridge, C.; Avis, J. M.; Nagai, K. J. Mol.
Biol. 1995, 249, 398–408.
Org. Lett., Vol. XX, No. XX, XXXX
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of the analogue with an ethylene glycol linkage was as high
as that of U1 snRNA I with an m32,2,7G cap, as shown in
Figure 5d, though the binding affinity of the U1 snRNA II
with a pyrophosphate linkage was lower than that of
U1 snRNA I. Ficner et al. previously reported that the
m32,2,7G interacts with another nucleobase and Trp276
of SNP1 in the crystal structure of the complex formed
between SPN 1 and the m32,2,7GpppG dimer.6e Therefore,
the shortage of the atom length of the pyrophosphate
linkage in U1 snRNA II might decrease the binding
affinity to SPN 1. On the other hand, a longer linkage
such as that afforded by ethylene glycol in U1 snRNA III
might not affect the binding affinity to SPN 1.
In summary, we carried out a first chemical synthesis of
U1snRNA and its analogues by a combination of solid-
phase and enzymatic reactions. The synthesis of U1
snRNA III with an m32,2,7G cap analogue was easier and
more efficient than that of U1 snRNA I with an m32,2,7G
cap. Moreover, we found that the binding affinity of U1
snRNA III was as high as that of U1snRNA I. These
results should be very useful for investigating splicing
correction and U1i by an artificial U1 snRNA in gene
therapy. Further studies on these issues are currently in
progress.
Figure 5. Electrophoretic mobility shift assay of the complexes
formed between snurportin 1 (0ꢀ2 μM) and U1 snRNA I-III
(50 nM) on a 16% nondenaturing polyacrylamide gel at pH 7.4.
(a) U1snRNA without an m32,2,7G cap, (b) unmodified U1
snRNA I, (c) modified U1 snRNA II, and (d) modified U1
snRNA III with an ethylene glycol linkage.
results are the first demonstration of the chemical synthesis
of U1 snRNA and their analogues.
Moreover, we examined the binding affinities of U1
snRNA IꢀIII to the nuclear import adaptor SPN 1, as
shown in Figure 5. The RNA without an m32,2,7G cap
could not specifically bind to SPN 1, though nonspecific
binding was observed in 2 μM SPN1 due to electrostatic
interaction between the phosphate groups of the RNA and
lysine (or arginine) residues of SPN1 (Figure 5a). On the
other hand, U1 snRNA I with an m32,2,7G cap could
completely bind to SPN1 in 1 μM SPN1 in Figure 5b.
Figure 5c and 5d show the results of U1snRNAs with
m32,2,7G cap analogues. Interestingly, the binding affinity
Acknowledgment. This study was supported by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan and Izumi Science and Technology Foundation.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data for all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
D
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