Chemical synthesis of site-specifically 2'-azido-modified RNA and potential applications for bioconjugation and RNA interference

Full text of DOI:10.1002/cbic.201000646

DOI: 10.1002/cbic.201000646
Chemical Synthesis of Site-Specifically 2’-Azido-Modified RNA and Potential
Applications for Bioconjugation and RNA Interference
Michaela Aigner,[a] Markus Hartl,[b] Katja Fauster,[a] Jessica Steger,[a] Klaus Bister,[b] and Ronald Micura*[a]
Dedicated to Prof. Albert Eschenmoser on the occasion of his 85th birthday.
One of the most important scientific discoveries in the recent
past concerns RNA interference (RNAi), which is a post-tran-
scriptional gene-silencing mechanism induced by small inter-
fering RNA (siRNA) and micro-RNA (miRNA).[1] RNAi has opened
up new avenues in the development of siRNA and miRNA as
therapeutic agents for various diseases.[2] The reason for the
large number of reports about chemically modified siRNA is
their potential to enhance nuclease resistance, to prevent
immune activation, to decrease off-target effects, and to im-
prove pharmacokinetic and pharmacodynamic properties, all
of which are important for the application of siRNA as thera-
peutic agents.[3] Another substantial challenge is siRNA deliv-
ery, because these reagents cannot easily traverse cell mem-
branes because of their size and negative charge.[4] To date,
the most promising therapeutic approach based on RNAi in-
volves chemically modified siRNA that can resolve some of the
issues mentioned above.
RNA double helix. Interestingly, this modification has not yet
been explored for siRNA technologies. It is even more surpris-
ing that, to the best of our knowledge, the solid-phase chemi-
cal synthesis of 2’-azido-modified RNA has not yet been de-
scribed.[8] The prospect of potential siRNA applications, and
also of promising applications in modern bioconjugation
chemistry (such as Staudinger ligation and click chemistry)[9]
prompted us to take up the challenge of synthesizing these
RNA derivatives (Scheme 1).
Chemical siRNA modifications belong to four classes—back-
bone, ribose, nucleobase, and terminal modifications—with
ribose modifications being the most common.[2b] Structurally
simple alterations, such as 2’-OCH3 and 2’-F, lead to significant-
ly enhanced performance of siRNA with diverse target genes,
provided that they are positioned in a site-specific manner.[3] In
particular, 2’-F modifications possess the extraordinary proper-
ty of being very well accepted onto the guide (antisense)
strand, while most other modifications are much better tolerat-
ed by the passenger (sense) strand.[5] The guide strand is incor-
porated into the crucial functional particle, the RNA-induced
silencing complex (RISC); thus RNA recognition and discrimina-
tion from non-native counterparts is very stringent.[6]
Scheme 1. Solid-phase chemical synthesis of RNA with site-specifically 2’-
azido-modified nucleosides, and the potential of these novel RNA derivates
for bioconjugation reactions and siRNA technologies.
Nucleosides that carry an azido group—no matter at which
position—cannot be isolated in the form of stable phosphora-
midite building blocks, the most convenient form for use in
advanced RNA solid-phase synthesis approaches.[10] This is due
to the inherent reactivity between phosphor-III species and
azides according to the Staudinger reaction. However, we had
experimental indications that this reactivity becomes prevalent
only at very high concentrations of azido-modified nucleoside
phosphoramidites (e.g., upon evaporation of the solvents to
isolate these compounds). This led us to consider the possibili-
ty that strand assembly by phosphoramidite chemistry might
still be feasible (at reasonable phosphoramidite concentra-
tions) once the azide moiety has been incorporated into the
RNA. Additionally, two very recent independent studies, one
on the chemical synthesis of 4’-azidomethyl-thymidine modi-
fied DNA and the other on the circularization of DNA by using
a solid-support bearing an azido linker, encouraged us to
target 2’-azido-RNA synthesis.[11a,b]
We postulated that siRNA with specific 2’-azido groups (2’-
N3) should have the potential for enhanced performance, be-
cause this functional group is small, polar, and supports the
C3’-endo ribose pucker[7] that is characteristic for an A-form
[a] M. Aigner, K. Fauster, J. Steger, Prof. Dr. R. Micura
Institute of Organic Chemistry
Center for Molecular Biosciences CMBI, University of Innsbruck
6020 Innsbruck (Austria)
Fax: (+43)507-512-2892
E-mail: ronald.micura@uibk.ac.at
We decided to focus on the 2’-azido-2’-deoxyuridine and the
2’-azido-2’-deoxyadenosine building blocks as 2-chlorophenyl
phosphodiester derivatives, 1 and 2, with the aim of employ-
ing them later in a single cycle of standard RNA phosphotri-
ester coupling for the incorporation into RNA, while strand as-
sembly should follow standard phosphoramidite chemistry.
For building block 1, we started the synthesis with 2,2’-anhy-
drouridine 3 (Scheme 2) which is commercially available or can
[b] Prof. Dr. M. Hartl, Prof. Dr. K. Bister
Institute of Biochemistry
Center for Molecular Biosciences CMBI, University of Innsbruck
6020 Innsbruck (Austria)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000646.
Re-use of this article is permitted in accordance with the Terms and Condi-
tions set out at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN) 1439–
4227/homepage/2268_onlineopen.html
ChemBioChem 2011, 12, 47 – 51
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
47

ether to form 9. Conversion into the corresponding phospho-
diester 2 was achieved in good yield by treatment with 2-
chlorophenyl chlorophosphorotriazolide.
For the incorporation of the 2’-azido-modified nucleoside
phosphodiester building blocks 1 and 2 into RNA, we conduct-
ed strand assembly by automated standard RNA solid-phase
synthesis with 2’-O-[(Triisopropylsilyl)oxy]methyl (2’-O-TOM)-
protected nucleoside phosphoramidites[17] up to the position
of the intended azide modification. The synthesis was inter-
rupted after the detritylation step that liberated the terminal
5’-hydroxyl group. Coupling of the phosphodiester building
block, 1 or 2, was achieved by activation with 1-(mesitylene-2-
sulfonyl)-3-nitro-1,2,4-triazole (MSNT) by using two syringes at-
tached to the column for reagent delivery and mixing under
argon.[18] By using this simple setup (see the Supporting Infor-
mation), coupling yields higher than 95% were achieved. After
manual capping, strand elongation was continued by standard
automated RNA phosphoramidite chemistry. With sequences
of up to about 25 nucleotides, we had no indication (from in-
spection of oligonucleotide by-products by LC-ESI mass spec-
trometry; see also refs. [10b] and [11a,c]) that the ribose 2’-
azido group had reacted with the phosphoramidite moiety
under the coupling conditions. For deprotection, the oligoribo-
nucleotides containing 2’-azido groups were first treated with
syn-2-pyridine aldoxime/tetramethylguanidine in dioxane/
water to cleave the 2-chlorophenyl phosphate protecting
groups.[18] Then, standard deprotection conditions were ap-
plied by using CH3NH2 in ethanol/water followed by treatment
with 1m tetra-n-butylammonium fluoride (TBAF) in THF. After
purification by anion-exchange HPLC, the expected molecular
weights were confirmed by liquid chromatography ESI (LC-ESI)
mass spectrometry (Table 1, Figure 1).
Scheme 2. Synthesis of the 2’-azido-2’-deoxyuridine 3’-phosphodiester build-
ing block 1 for RNA solid-phase synthesis. Reagents and conditions:
a) 1.8 equiv LiF, 1.8 equiv (CH3)3SiN3, in N,N,N’,N’-tetramethylethylenediamine
(TMEDA)/DMF 1:1, 1108C, 48 h, 66%; b) 1.5 equiv 4,4’-dimethoxytriphenyl-
methyl chloride (DMT-Cl), in pyridine, RT, 24 h, 69%; c) i: 4 equiv N-methyl-
imidazole, in THF, RT, 5 min; ii: 2.5 equiv 2-chlorophenyl phosphorodichlori-
date, 5.5 equiv 1,2,4-triazole, 5 equiv Et3N, in THF, RT, 10 min, 86%.
be obtained from uridine by a single transformation by using
diphenyl carbonate in DMF.[12] Nucleophilic ring-opening with
in situ-generated lithium azide in DMF furnished the 2’-azido-
2’-deoxyuridine derivative 4.[13] Subsequently, the 5’-hydroxyl
group was protected as dimethoxytrityl (DMT) ether to form
compound 5.[14] Conversion into the corresponding phospho-
diester 1 was achieved in good yield by reaction with in situ-
generated 2-chlorophenyl chlorophosphorotriazolide, analo-
gously to a general procedure in the literature.[15]
For building block 2, we started the synthesis with the tri-
flated adenosine derivative 6, which was readily obtained from
adenosine according to the previously described synthesis to
2’-methylseleno adenosine (Scheme 3).[16] Treatment of 6 with
lithium azide in DMF furnished key compound 7, followed by
cleavage of the silyl protecting group to give the diol deriva-
tive 8. The 5’-hydroxyl group was then protected as the DMT
Table 1. Selection of synthesized 2’-azido-2’-deoxyuridine (U*) and 2’-
azido-2’-deoxyadenosine (A*) containing oligoribonucleotides.
Sequence (5’!3’)
Amount
[nmol]
MW calcd
[amu]
MW obs
[amu]
GGU*CGACC
GGUCGA*CC
CCAGGCCU*GG
CCA*GGCCUGG
GAAGGGCAACCU*UCG
GAA*GGGCAACCUUCG
G2UCUCU*GCCA2UA2GACATT
UGUCU2A*U2G2CAGAGAC2TdG
UGUCU*UAUU*G2CAGAGAC2TdG 55
UGUCU2AUU*G2CA*GAGAC2TdG 68
125
196
87
2549.6
2549.6
3200.1
3200.1
4839.0
4839.0
6678.1
6697.1
6722.1
6722.1
2549.2
2549.2
3200.2
3200.2
4838.4
4838.3
6677.8
6696.8
6721.5
6721.2
79
173
161
298
120
Next we investigated the influence of the 2’-azido group on
the thermal stability of RNA double helices by using tempera-
ture-dependent UV spectroscopy (Figure 2A). At 150 mm NaCl
and 10 mm Na2HPO4 (pH 7.0), the unmodified hairpin 5’-
GAAGGGCAACCUUCG melted at 72.5ꢀ0.58C while the 2’-
azido-modified counterparts, 5’-GAAGGGCAACCU*UCG and 5’-
GAA*GGGCAACCUUCG, displayed Tm values of 71.7ꢀ0.58C and
71.6ꢀ0.58C, respectively. This demonstrates that the 2’-azido
Scheme 3. Synthesis of the 2’-azido-2’-deoxyadenosine 3’-phosphodiester
building block 2 for RNA solid-phase synthesis. Reagents and conditions:
a) 5 equiv LiN3, in DMF, RT, 20 h, 86%; b) 1m TBAF, 0.5m CH3COOH, in THF,
RT, 2 h, 71%; c) 1.5 equiv DMT-Cl, in pyridine, RT, 16 h, 64%; d) i: 4 equiv N-
methylimidazole, in THF, RT, 5 min; ii: 2.5 equiv 2-chlorophenyl phosphorodi-
chloridate, 5.5 equiv 1,2,4-triazole, 5 equiv Et3N, in THF, RT, 10 min, 79%.
48
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ChemBioChem 2011, 12, 47 – 51

Figure 1. Characterization of 2’-azido-modified oligoribonucleotides by
anion-exchange HPLC and LC-ESI mass spectrometry. A) 8 nucleotide (nt)
RNA containing 2’-azido-2’-deoxyadenosine. B) 10 nt RNA containing 2’-
azido-2’-deoxyuridine. C) 15 nt RNA containing 2’-azido-2’-deoxyadenosine.
For conditions see the Supporting Information.
Figure 3. Attachment of a fluorescence quencher to RNA with an internal
site-specific ribose 2’-azido group. A) Concept of molecular beacon (MB);
B) Staudinger ligation of the phosphine derivative 10 to RNA 11; C) Time-
dependent fluorescence response of the MB 12 to addition of the target se-
quence 13 (left) and of the near-cognate sequence 14 (right) with double
mismatch discrimination (mismatched nucleobases underlined). Conditions:
cMB =0.5 mm, ctarget =2.5 mm, 100 mm MES·NaOH, 1.0 mm MgCl2, pH 6.5.
lex =490 nm, lem =514 nm. f=fluorescence (emission), t=time.
and Q and thus fluorescence emission. Here we wished to
demonstrate that the attachment of a dye to the 2’-azido posi-
tion can be performed post-synthetically by following one of
the most widespread bioconjugation strategies, the Staudinger
ligation.[9a,20] We synthesized the Disperse Red 1 (DR1) phos-
phine derivative 10 (see Supporting Information) and then ap-
plied typical Staudinger conditions[20] in H2O/DMF for ligation
to the 5’-fluorescein-labeled RNA 11 (Figure 3B). With a 100-
fold excess of 10, we observed complete conversion of 11 into
the amide- (12) and the O-methyl imidate (12a) linked DR1-
RNA conjugates (85%), and a minor amount (15%) of the
reduced (nonconjugated) 2’-NH2 RNA derivative of 11 (see the
Supporting Information).
Figure 2. Spectroscopic characterization of 2’-azido-modified oligoribo-
nucleotides. A) Overlay of UV melting profiles of GAAGGGCAACCUUCG,
GAAGGGCAACCU*UCG, GAA*GGGCAACCUUCG, cRNA =5 mm; B) Overlay of CD
spectra of GAAGGGCAACCUUCG, GAAGGGCAACCU*UCG, GAA*GGGCAAC-
CUUCG, cRNA =2 mm. Conditions: 10 mm Na2HPO4, 150 mm NaCl, pH 7.0.
U*=2’-azido-2’-deoxyuridine, A*=2’-azido-2’-deoxyadenosine, H=hyper-
chromicity.
group is modestly destabilizing, which might arise from a
slight steric interference of the moiety with the 3’-phos-
phate.[7a] Additionally, we examined this hairpin system with
CD spectroscopy: the spectra clearly indicate that the overall
conformation of a typical A-form double helical geometry is re-
tained in the 2’-azido-modified RNA (Figure 2B).
To assess the potential of site-specifically 2’-azido-modified
RNA for bioconjugation reactions[9] we attached a fluorescence
quencher to the azido group to construct a molecular beacon
(MB).[19] MBs are widely used probes for the specific detection
of DNA and RNA targets, and their function is based on the
interaction between a fluorescent (F) and a quenching (Q)
moiety (Figure 3A). In the absence of the target, the MB
adopts a hairpin structure that brings F and Q into close prox-
imity resulting in quenching of fluorescence. In contrast, hy-
bridization with the target of the MB leads to separation of F
This clearly shows that, despite the relatively high steric hin-
drance of the ribose 2’-azido group in RNA, its reactivity is still
sufficient for efficient chemical ligation. Moreover, the fluores-
cence responses of 12 depicted in Figure 3C demonstrate the
expected functionality for target recognition and for discrimi-
nation from near-cognate sequences.
In order to evaluate the potential of 2’-azido-modified oli-
goribonucleotides in siRNA applications, we employed a model
system for the knockdown of the brain acid soluble protein 1
(BASP1) gene by transient siRNA nucleofection in the chicken
DF-1 cell line by using standard commercial siRNA duplexes as
previously described.[21a] Expression of the BASP1 gene is spe-
cifically suppressed by the Myc oncoprotein, while the BASP1
protein is an efficient inhibitor of Myc-induced cell transforma-
ChemBioChem 2011, 12, 47 – 51
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49

tion.[21a] We synthesized ten siRNA duplexes for the Myc target
BASP1 with a single 2’-azido-2’-deoxyuridine or 2’-azido-2’-de-
oxyadenosine modification and the sequence organisation de-
picted in Figure 4A (see also Supporting Information). All the
modifications (2’-F and 2’-OCH3) that are excellently tolerated
in the guide strand.[3d,5,6a,22]
In conclusion, the solid-phase synthesis approach presented
here efficiently yields RNA with site-specific 2’-azido groups,
and provides a reliable foundation for a wide range of applica-
tions in modern bioconjugation strategies and siRNA technolo-
gies. Clearly, to evaluate the full potential of this type of modi-
fication, systematic studies of 2’-azido-modified siRNA on nu-
clease resistance, immune activation, off-target effects, toxicity,
membrane permeability, and delivery will be required. We are
confident that, in particular, the small size and the high polari-
ty of the azido group will provide improvements for at least
some of these effects. Moreover, the 2’-azido group in siRNA is
available for the attachment of hydrophobic tags (such as cho-
lesterols, lipids, cofactors, cell-penetrating peptides) to en-
hance delivery properties. Additionally, the attachment of fluo-
rescent dyes to the azido anchor (as exemplified here in the
context of MBs) facilitates the localization and tracking of
siRNA in the cell. Another feature that has to be explored in
future studies of 2’-azido-RNA (and in particular of 2’-azido-
siRNA) is photoreactivity, and whether it can be exploited for
crosslinking to peptide and protein targets. All these aspects
represent the focus of ongoing studies in our laboratory to-
gether with the expansion of the concept towards 2’-azido-2’-
deoxycytidine- and 2’-azido-2’-deoxyguanosine-containing
RNA. At the current state of research, the 2’-azido modification
might prove superior to the more than 35 other types of
chemical modifications that have been tested for their effects
on siRNA performance.[2] It might also prove useful in the ma-
nipulation of other types of noncoding RNA in the cell.[23]
Figure 4. Gene silencing by 2’-azido-2’-deoxyuridine- and 2’-azido-2’-deoxya-
denosine-modified siRNA. A) Sequence of the BASP1 siRNA used in this
study;[21a] nucleosides in red indicate positions of 2’-azido modification.
B) Biological activities of 2’-azido siRNAs directed against BASP1 mRNA.
Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected
with 0.12 nmol (~1.5 mg) aliquots of unmodified commercial (Qiagen) or
own (unmod) synthetic siRNAs (SIR) directed against the chicken BASP1
mRNA, or of siRNAs containing azido (Az) groups at the indicated nucleo-
tides on sense (s) or antisense (as) strands. An equal aliquot of siRNA with a
shuffled (random) nucleotide sequence was used as a control. Total RNAs
were isolated two days after siRNA delivery and 5 mg-aliquots were analyzed
by Northern hybridization by using DNA probes specific for the chicken
BASP1 gene, or the quail housekeeping GAPDH gene. RNAs (5 mg) from
normal quail embryo fibroblasts (QEF), and from QEF transfected with the
retroviral construct RCAS-v-myc[21b] encoding the BASP1-suppressing v-Myc
oncoprotein were used as controls. The electrophoretic positions of riboso-
mal RNA are indicated in the margin. All siRNAs depicted contain overhangs
of 2’-deoxynucleosides (lower case letters). SIR random: 5’-UCUGGGU-
CUAAGCCAAACAUT/5’-UGUUUGGCUUAGACCCAGAUdG.
Acknowledgements
M.A. thanks the Austrian Academy of Science and UNESCO for a
l’Orꢀal fellowship. Funding by the Austrian Science Foundation
FWF (P21641, I317 to R.M.; P18148 to M.H.; P17041 to K.B.) and
the Ministry of Science and Research (GenAU III, “non-coding
RNAs” P0726-012-012 to R.M.) is acknowledged. M.A. and R.M.
thank Claudia Hçbartner (MPI Gçttingen) for stimulating discus-
sions and critical comments on the manuscript.
Keywords: azides
·
gene
silencing
·
labeling
·
phosphoramidite · siRNA · solid-phase synthesis
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