2′-azido RNA, a versatile tool for chemical biology: Synthesis, X-ray structure, siRNA applications, click labeling

Article abstract of DOI:10.1021/cb200510k

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks an

Full text of DOI:10.1021/cb200510k

Articles
pubs.acs.org/acschemicalbiology
2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray
Structure, siRNA Applications, Click Labeling
Katja Fauster,† Markus Hartl,‡ Tobias Santner,† Michaela Aigner,† Christoph Kreutz,† Klaus Bister,*,‡
Eric Ennifar,*,§ and Ronald Micura*,†
‡
†Institute of Organic Chemistry and Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of
Innsbruck, 6020 Innsbruck, Austria
§Architecture et Rea
́ ́ ́ ́
ctivite de l′ARN, Institut de Biologie Moleculaire et Cellulaire, CNRS/Universite de Strasbourg, 67084
Strasbourg, France
S
* Supporting Information
ABSTRACT: Chemical modification can significantly enrich
the structural and functional repertoire of ribonucleic acids and
endow them with new outstanding properties. Here, we report
the syntheses of novel 2′-azido cytidine and 2′-azido guanosine
building blocks and demonstrate their efficient site-specific
incorporation into RNA by mastering the synthetic challenge
of using phosphoramidite chemistry in the presence of azido
groups. Our study includes the detailed characterization of 2′-
azido nucleoside containing RNA using UV-melting profile
analysis and CD and NMR spectroscopy. Importantly, the X-
ray crystallographic analysis of 2′-azido uridine and 2′-azido
adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The
2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the
minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken
fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site.
Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of
rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The
latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent
labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including
all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far
neglected type of RNA modification.
peptides, lipids or cofactors) or labeling with fluorescence dyes
to track the siRNAs in the cell. Both types of conjugates are
accessible via modern bioorthogonal conjugation reactions.11,12
In a recent communication, we have originally reported on
2′-azido modified RNA.13 Surprisingly, this modification had
previously been neglected in the context of siRNA, most likely
because of expected synthetic difficulties14 with standard solid-
phase RNA phosphoramidite chemistry based on the inherent
reactivity between phosphor-III species and azides. Never-
theless, the prospect of possible siRNA applications,6 but also
of promising applications in modern bioconjugation chem-
istry,11,12 prompted us to target 2′-azido RNA. We synthesized
phosphodiester building blocks of 2′-azido-2′-deoxyuridine and
2′-azido-2′-deoxyadenosine and demonstrated that their cou-
pling under standard conditions of RNA phosphotriester
chemistry together with standard phosphoramidite chemistry
he structural and functional repertoire of ribonucleic acids
T(RNA) can be significantly manipulated by chemical
modifications. This is of particular importance for two major
tools in current RNA research, namely, RNA interference
(RNAi) and RNA bioconjugation. RNAi is a post-transcrip-
tional gene silencing mechanism induced by small interfering
RNA (siRNA) and micro-RNA (miRNA).1,2 It has become one
of the major tools for gene function analysis and has also been
confronted with high expectations for the development of
siRNA and miRNA as therapeutic agents to treat diseases.3,4
However, for applications of siRNA as therapeutic agents,
chemical modification is obligatory to enhance nuclease
resistance, to prevent immune activation, to decrease off-target
effects, and to improve pharmacokinetic and pharmacodynamic
properties.5−9 Additionally, the modifications should help these
agents to penetrate cell membranes and improve siRNA
delivery which remains one of the major challenges.10 This
latter issue closes the circle from siRNAs to bioconjugation
since many approaches require an attachment of the nucleic
acid portion to transporter units of diverse structure (such as
Received: December 4, 2011
Accepted: January 9, 2012
Published: January 24, 2012
© 2012 American Chemical Society
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for assembly of the other nucleotides works sufficiently well to
achieve these RNA derivatives.13
Here, we present the synthesis of the novel phosphodiester
building blocks of 2′-azido-2′-deoxycytidine 7 and 2′-azido-2′-
deoxyguanosine 17 and expand the site-specific introduction of
the 2′-azido modification into RNA to all four canonical
nucleosides (Figure 1). Our study includes the detailed
triisopropylbenzenesulfonyl chloride in the presence of triethyl-
amine and DMAP in dichloromethane resulted in regioselective
O4-trisylation. After workup, the trisylated derivative 3 can be
used without further purification and directly converted into 4
upon treatment with aqueous ammonium hydroxide in THF in
80% yield over the two steps (which is significantly higher
compared to when compound 3 is isolated and purified by
column chromatography). We also note that we applied
aqueous ammonium hydroxide conditions based on our
experience from a previous study where treatment of the 2′-
methylseleno analog of 3 with 7 M NH3 in anhydrous methanol
resulted in the corresponding O4-methyluridine derivative.15
Acetylation of the amino function was then achieved with acetic
anhydride in pyridine to provide 5, followed by cleavage of the
3′-O-TBDMS group with 1 M TBAF and 0.5 M acetic acid in
THF to give 6. Finally, conversion into the corresponding
phosphodiester 7 was achieved in good yields by reaction with
in situ generated 2-chlorophenyl chlorophosphorotriazolide in
analogy to a general procedure in the literature.17 Starting with
compound 1, our route provides 7 in a 43% overall yield in six
steps with four chromatographic purifications; in total, 2.1 g of
7 was obtained in the course of this study.
For building block 17 (Scheme 2), our route began with the
tetraisopropyldisiloxane (TIPDS) 3′- and 5′-protected guano-
sine derivative 8,16 followed by protection of the exocyclic
guanine 2-amino group using N,N-dimethylformamide dimeth-
yl acetal to furnish derivative 9 and protection of the guanine
lactam moiety with a O6-(4-nitrophenyl)ethyl group introduced
under Mitsunobu conditions to give 10. Then, triflation of the
ribose 2′-OH resulted in intermediate 11, which was converted
into the arabino nucleoside 12 in diastereoselective manner by
treatment with potassium trifluoroacetate and 18-crown-6-
ether. After triflation of the arabinose 2′-OH, compound 13 was
reacted with lithium azide, producing key derivative 14 in high
yields. Deprotection of the TIPDS moiety proceeded
Figure 1. Structure of RNA with site-specifically 2′-azido-modified
nucleosides. Such derivatives carry high potential for bioconjugations
and applications in RNA interference.
characterization of the 2′-azido modified RNA using UV, CD,
and NMR spectroscopy. Importantly, we have solved the X-ray
structures of 2′-azido containing model double helices at atomic
resolution and we performed a series of experiments to evaluate
their siRNA performance and potential for labeling using Click
chemistry.
RESULTS AND DISCUSSION
■
Synthesis of 2′-Azido Nucleoside Building Blocks. For
building block 7 (Scheme 1), we started the synthesis from the
2′-azido-2′-deoxyuridine derivative 1,13 which was readily
obtained from uridine. The 3′-OH of compound 1 was
protected applying TBDMS chloride and imidazole in DMF
to furnish derivative 2. Then, reaction of 2 with 2,4,6-
a
Scheme 1. Synthesis of 2′-Azido Cytidine Building Block 7
a
Reaction conditions: (a) 2.0 equiv TBDMSCl, 4.0 equiv imidazole, in DMF, RT, 16 h, 95%; (b) 1.5 equiv 2,4,6-triisopropylbenzenesulfonyl
chloride, 10.0 equiv NEt3, 0.12 equiv DMAP, in CH2Cl2, RT, 1 h, 60%; (c) 32% aqueous NH3, in THF, RT, 16 h, 95%; (d) 2.5 equiv acetic
anhydride, in pyridine, 0°C to RT, 90 min, 97%; (e) 1 M TBAF/0.5 M acetic acid, in THF, RT, 2.5 h, 100%; (f) 5.5 equiv 1,2,4-triazole, 5.0 equiv
NEt3, 2.5 equiv 2-chlorophenyl phosphorodichloridate, 4.0 equiv 1-methylimidazole in THF, RT, 1 h, 82%.
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a
Scheme 2. Synthesis of 2′-Azido Guanosine Building Block 17
a
Reaction conditions: (a) 2.5 equiv N,N-dimethylformamide dimethyl acetal, in DMF, 6 h, RT, 84%; (b) 1.3 equiv NPE-OH, 1.4 equiv PPh3, 1.3
equiv DIAD, in dioxane, 16 h, RT, 40%; (c) 1.5 equiv trifluoromethanesulfonyl chloride, 3.0 equiv DMAP, in CH2Cl2, 30 min, 0°C, 44%; (d) 5.0
equiv CF3COO−K+, 2.0 equiv 18-crown-6, 1.5 equiv (iPr)2NEt3 in toluene, 16 h, 80°C, 99%; (e) 1.5 equiv trifluoromethanesulfonyl chloride, 2.5
equiv NEt3, 1.5 equiv DMAP, in CH2Cl2, 15 min, 0°C, 82%; (f) 5.0 equiv LiN3, in DMF, 16 h, RT, 71%; (g) 1 M TBAF/0.5 M AcOH, in THF, 2.5
h, RT, 93%; (h) 1.4 equiv DMT-Cl, in pyridine, 16 h, RT, 91%; (i) 5.5 equiv 1,2,4-triazole, 5.0 equiv NEt3, 2.5 equiv 2-chlorophenyl
phosphorodichloridate, 4.0 equiv 1-methylimidazole in THF, RT, 1 h, 70%.
straightforward using tetrabutylammonium fluoride (TBAF)
and acetic acid. Finally, derivative 15 was transformed into the
dimethoxytritylated compound 16, and conversion into the
corresponding phosphodiester 17 was achieved in good yields
by reaction with in situ generated 2-chlorophenyl chlorophos-
phorotriazolide in analogy to a general procedure in the
literature.17 Starting with compound 8, our route provides 17 in
a 5% overall yield in nine steps with seven chromatographic
purifications; in total, 0.5 g of 17 was obtained in the course of
this study. A recent report on the synthesis of 2′-
methylselenoguanosine18 suggested that protection of O6
could be omitted to shorthen the synthesis of 17; however,
in our hands, we observed significant triflation at O6 when the
lactam moiety was unprotected.
Synthesis of 2′-Azido Modified RNA. For the incorpo-
ration of the 2′-azido-modified nucleoside phosphodiester
building blocks 7 and 17 into RNA, we conducted strand
assembly by automated standard RNA solid-phase synthesis
using 2′-O-TOM protected nucleoside phosphoramidites19 up
to the position of the intended azide modification. The
synthesis was interrupted after the detritylation step that
liberated the terminal 5′-hydroxyl group. Then, coupling of the
phosphodiester building block 7 or 17 was achieved manually
by activation with 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-tria-
zole (MSNT)20 in yields up to 95%. After capping, strand
elongation was continued by automated RNA phosphoramidite
chemistry. Up to an elongation of about 25 nucleotides we had
no indication (from inspection of oligonucleotide byproduct by
LC−ESI mass spectrometry) that the ribose 2′-azido group
reacted with the phosphoramidite moiety under the coupling
conditions used. For deprotection, the oligoribonucleotides
containing 2′-azido groups were first treated with syn-2-pyridine
aldoxime/tetramethylguanidine in dioxane/water to cleave the
2-chlorophenyl phosphate protecting groups.20 Then, standard
deprotection conditions using CH3NH2 in ethanol/water
followed by treatment with 1 M TBAF in THF were applied.
We point out that the O6-(4-nitrophenyl)ethyl groups are
stable during the first deprotection step (CH3NH2 in ethanol/
water) but become cleaved during the second step of
deprotection (TBAF in THF) when fluorine anions induce β-
elimination to release the lactam moieties of the guanine
nucleobases.21−23 After purification by anion-exchange HPLC
(Supporting Figure 1), the expected molecular weights were
confirmed for all RNAs by LC−ESI mass spectrometry (Table
1, Supporting Figure 1).
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a
Table 1. Selection of 2′-Azido Modified RNAs
molecular weight
[amu]
yield
[nmol]
sequence (5′→3′)
calcd
obsd
GGCN3UAGCC
90
159
26
2549.6
4838.9
6678.1
6678.1
6697.1
6697.1
2549.6
6697.1
6697.1
6697.1
2550.0
4838.9
6677.8
6677.4
6696.7
6697.2
2549.6
6696.5
6696.5
6696.6
GAAGGGCAACCN3UUCG
GGUCUCUGCCN3AAUAAGACATT
GGUCUCUGCN3CAAUAAGACATT
UGUCUUAUUGGCN3AGAGACCTdG
UGUCUUAUUGGCAGAGACCN3TdG
GGCUAGN3CC
UGUCUUAUUGN3GCAGAGACCTdG
UGUCUUAUUGGN3CAGAGACCTdG
UGUCUUAUUGGCAGN3AGACCTdG
15
332
394
110
90
42
Figure 2. 1H NMR imino proton spectra of 2′-azido-modified
oligoribonucleotides. (A) GGCUAGCC (top) and GGCUAGN3CC
(bottom). (B) GAAGGGCAACCUUCG (top) and GAAGGG-
CAACCN3UUCG (bottom); conditions: cRNA = 0.1 mM, 25 mM
Na2HAsO4, pH 7.0. CN3, 2′-N3 cytidine; GN3, 2′-N3 guanosine.
10
a
CN3, 2′-N3 cytidine; GN3, 2′-N3 guanosine
Pairing Stability of 2′-Azido Modified RNA. Next, we
investigated the influence of the 2′-azido group on the thermal
stability of RNA double helices by temperature-dependent UV
spectroscopy (Supporting Figure 2). At 150 mM NaCl and 10
mM Na2HPO4 (pH 7.0), the non-modified, self-complemen-
X-ray Analysis of a 2′-Azido Containing RNA.
Encouraged by the NMR spectroscopic results, we set out for
the X-ray analysis of a 2′-azido modified RNA. We focused on
the 27 nt fragment of E. coli 23 S rRNA sarcin-ricin loop (SRL)
region25,26 as a crystallization scaffold. The modification of
interest was placed in the double helical region of this scaffold
to obtain insights on the impact of a 2′-azido group within an A-
form RNA double helix. Analysis of the SRL structure (Protein
Data Bank [PDB] identification no. 483D) revealed that the 2′-
OH groups of U2650 and A2670 are not involved in crystal
contacts and hence should be available for modifications.
Moreover, these two nucleotides are forming a Watson−Crick
base pair in a double helical region. Three SRL sequences were
therefore synthesized: an unmodified SRL (used as a control
for crystallization and diffraction) and two modified sequences
with the introduction of a 2′-azido group into U2650 or A2670
(Figure 3, Supporting Figure 5 and Supporting Figure 6).
Crystallization trials revealed that both 2′-azido modified RNAs
crystallized, providing crystals diffracting to atomic resolution
(Supporting Table 2). X-ray structure determinations showed
that the azido groups are well-defined in the electron density
maps for both 2′-azido modified RNAs (Figure 3). Super-
impositions of both azido-modified RNA structures with the
unmodified RNA revealed a root-mean-square deviation (rmsd)
of 0.07 and 0.14 Å. These values were within the errors on
coordinates (0.13 and 0.11 Å), thus showing that the 2′-azido
group does not affect the RNA structure (Figure 4). However,
detailed analysis of the RNA hydration pattern disclosed a
displacement of several water molecules from the RNA major
groove in presence of the 2′-azido group (Figure 4B,C). In
addition, a water molecule appears to be involved into an
intraresidue U(O2)/water/2′-azido interaction in the 2′-N3-
U2650 structure (Figure 3B). These modifications of the RNA
hydration shell might be responsible for the slight changes in
melting temperatures compared to the unmodified RNA.
Additionally, another possible contribution of the decrease in
thermal stability could also be a small, but significant, influence
of the azido group on the nucleobase polarization, as recently
postulated for a 2′-F-modified RNA.27
tary RNA 5′-GGCUAGCC-3′ melted at 62.2 0.5 °C (cRNA
=
16 μM), while the 2′-azido guanosine modified counterpart 5′-
GGCUAGN3CC-3′ displayed a Tm value of 59.3 0.5 °C (cRNA
= 16 μM) (Supporting Figure 2A). This demonstrated that the
2′-azido group causes a slight decrease in thermal stability,
about −1.4 °C per modification since the influence stems from
two isolated 2′-N3-G/C base pairs (bp) within the palindromic
duplex. A detailed determination of the thermodynamic
stabilities based on the concentration dependence of their
melting points24 provided a ΔG° of −13.7 0.3 kcal/mol for
the unmodified duplex and −13.3 0.3 kcal/mol for the 2′-N3-
G containing duplex (Supporting Figure 3). Additionally, we
evaluated the influence on the thermal stability of the
corresponding 2′-azido cytidine nucleoside and therefore
compared the hairpin forming RNA 5′-GAAGGGCAAC-
CUUCG to its 2′-azido cytidine modified counterpart 5′-
GAAGGGCAACCN3UUCG. While the non-modified RNA
melted at 71.7
minimally increased Tm value of 72.3
0.5 °C, the modified hairpin showed a
0.5 °C (Supporting
Figure 2C). As expected for a monomolecular melting
transition, the melting points did not change in response to
varying concentration (cRNA = 2−32 μM). Shape analysis of the
melting curves24 provided the thermodynamic stabilities of a
ΔG° of −6.6 0.3 kcal/mol for the unmodified duplex and
−6.5
0.3 kcal/mol for the 2′-N3-C containing duplex
(Supporting Figure 4).
CD and NMR Spectroscopic Analysis. We applied CD
spectroscopy to the duplex and hairpin systems, and the spectra
clearly indicated that the overall conformation of a typical A-
form double helical architecture was retained for the RNA
double helices with 2′-azido-C or 2′-azido-G containing base
pairs (Supporting Figure 2B,D).
1
The H NMR spectra of the duplex and hairpin systems
described above were recorded, and we observed that the
chemical shift as well as the line shape of the imino proton
resonances were again hardly affected by the modification
(Figure 2). As these resonances reflect the integrity of
Watson−Crick base pairs, the H NMR data suggests that 2′-
azido-C or 2′-azido-G containing base pairs are well tolerated
RNA Interference of 2′-Azido Modified siRNA. We have
recently pointed at the promising potential of 2′-azido modified
RNA for RNA interference, exemplified by using siRNA
duplexes with single and double 2′-azido uridine and/or 2′-
1
within RNA double helices in aqueous buffer solution.
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(Figure 5B), with the highest remaining level of expression of
about 10% observed for “SIR Az-G14 as”. Remarkably, the 2′-
azido group is very well tolerated in the guide strand, even
when the site of modification is directly at the cleavage site
(positions 10, 11). As expected, the modification caused
increased nuclease resistance (Supporting Figure 7). Moreover,
we compared the 2′-azido modified siRNAs to their 2′-fluoro
and 2′-O-methyl counterparts and found comparable silencing
efficiencies (Figure 5C). Additionally, we mixed 2′-azido, 2′-
fluoro and 2′-O-methyl modifications up to a number of seven
within the antisense strand (Figure 5C). Some of these
modified duplexes showed more reduced efficiencies and
remaining expression levels of about 25−35%, even for non-
azido siRNA species. Reduced efficiency was also observed for a
siRNA type with a total of two 2′-fluoro (positions 9, 11) and
three 2′-O-methyl groups (positions 3, 7, 15). When we added
two 2′-azido modifications to either positions 8 and 12 or 8 and
13, the expression levels were slightly increased, thereby less for
the first mentioned substitution pattern. In a further experi-
ment, we demonstrated that the level of suppression of these
highly modified siRNAs was dose-dependent (Supporting
Figure 8). Moreover, time-course experiments showed a
significant level of suppression even after 2 days of incubation
for the unmodified as well as the highly modified siRNA
duplexes, whereas after 9 days full expression levels were
reached again for all four duplexes tested (Supporting Figure
9). Taken together, our siRNA data demonstrates that the 2′-
azido group represents an interesting and powerful alternative
that complements the existing repertoire of 2′-modifications for
siRNA applications.
Labeling of 2′-Azido RNA: Click Chemistry. To further
underline the high chemical flexibility of 2′-azido modified RNA
strands, we demonstrated their amenability to one of the most
widely used bioconjugation reaction, namely ,the azide−alkyne
1,3-dipolar cycloaddition reaction, commonly referred to as
Click chemistry.11,12 We chose the copper-catalyzed version
with acetonitrile as cosolvent acting as ligand of the CuI
complex, thereby stabilizing the oxidation state.30 Applying
this setup, 2′-azido modified BASP1 siRNA at 1 mM
concentration was efficiently reacted with a commercially
available, alkyne-modified 5-carboxytetramethylrhodamine dye
(F545) (2 mM) in the presence of sodium ascorbate and
analyzed by anion exchange chromatography (Figure 6A,B).
While the siRNA labeled in standard fashion at the 3′-end of
the sense strand was of the same activity compared to the
Figure 3. X-ray structure of 2′-azido modified RNA at atomic
resolution. (A) E. coli Sarcin-ricin stem-loop (SRL) RNA used for
crystallization; secondary structure. (B) 2Fobs − Fcalc electron density
map showing the 2′-N3-U2650/A2670 base pair. Water molecules are
shown as red spheres. (C) 2Fobs − Fcalc electron density map showing
the U2650/2′-N3-A2670 base pair.
azido adenosine derivatizations.13 Here, we expand these
investigations toward 2′-azido cytidine and 2′-azido guanosine
to further evaluate 2′-azido-modified oligoribonucleotides for
siRNA applications. For reasons of comparability, we employed
the same model system used previously to knock down the
brain acid soluble protein 1 (BASP1) encoding gene by
transient siRNA nucleofection in the chicken DF-1 cell line.13,28
Expression of the BASP1 gene is specifically suppressed by
Myc, an evolutionary conserved oncoprotein;29 conversely, the
BASP1 protein is an efficient inhibitor of Myc-induced cell
transformation.28 First, we synthesized six siRNA duplexes for
the BASP1 target gene with the sequence organization depicted
in Figure 5A (see also Supporting Table 1), three of them with
a single 2′-azido-2′-deoxycytidine and three of them with a
single 2′-azido-2′-deoxyguanosine modification, all of the
modifications in either very close vicinity, or directly at the
cleavage site. The modified siRNAs caused significant gene
silencing as observed for the non-modified reference duplex
Figure 4. Structure comparison of 2′-azido modified and unmodified SRL RNA. (A) Stereoview showing a superposition of unmodified (gray), 2′-
N3-A2670 (red), and 2′-N3-U2650 (blue) RNA; arrows indicate positions of 2′-N3 groups. (B, C) Detailed views (two different orientations) of the
U2650-A2670 base pair and hydration in superposed RNA structures with and without 2′-N3 modification. A water molecule hydrogen-bonded with
2′-OH and O2 of U2650 is shifted in the 2′-N3-U2650 SRL structure (green arrow).
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Figure 5. Gene silencing by 2′-azido modified siRNAs. (A) Sequence of the brain acid soluble protein 1 (BASP1)13,28 targeting siRNA duplex used in
this study; nucleosides in red indicate positions for 2′-azido modification tested. (B) Biological activities of 2′-azido cytidine or 2′-azido guanosine
modified siRNAs. (C) Biological activities of 2′-OMe (green), 2′-F (blue), and 2′-N3 (red) modified siRNAs, directed against BASP1 mRNA.
Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected with 0.12 nmol (∼1.5 μg) aliquots of unmodified (SIR) or modified
siRNAs (SIR Az, SIR OMe, SIR F) containing azido, methoxy, or fluoro groups at the indicated nucleotides 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 2 days after siRNA
delivery, and 5-μg aliquots were analyzed by Northern hybridization using a DNA probe specific for the chicken BASP1 gene, and subsequently a
probe specific for the housekeeping quail GAPDH gene.28 The levels (%) of BASP1 expression were determined using a phosphorimager and are
depicted as bars relative to mock transfections (no SIR, 100%). The electrophoretic positions of rRNAs are indicated in the margin. All siRNAs
depicted contain overhangs of 2′-deoxynucleosides (lower case letters). SIR random: 5′-UCUGGGUCUAAGCCAAACAUT/5′-UGUUUGG-
CUUAGACCCAGAUdG.
unmodified reference, siRNAs with the fluorophore attached to
the antisense strand at internal nucleoside positions exhibited
reduced activities (Figure 6C). The latter is not unexpected due
to the stringent structural requirements for strand recognition
within the RISC complex.6 However, since a very recent study
has reported on successful examples of fluorescent labeling of
the antisense strand,31 we also evaluated our type of labeling for
three selected internal antisense positions. We observed
significant but reduced efficiencies for two of the corresponding
siRNAs (C19, G11), while the C12-labeled counterpart had less
activity (Figure 6C). Nevertheless, the fluorescently labeled
siRNAs allowed their reliable localization within the chicken
DF1 cells by fluorescence microscopy (Figure 6D). Taken
together, this set of experiments demonstrates that the 2′-azido
group of our RNA derivatives can efficiently react under Click
conditions and is therefore open to the wide field of
bioconjugation and applications.
Summary, Reflection, and Conclusion. This study
reports clear evidence of the accessibility of site-specifically
2′-azido modified RNA with respect to all four standard
nucleosides. Importantly, the azido nucleosides, once incorpo-
rated into RNA by individual cycles of phosphotriester
chemistry, are compatible with subsequent strand assembly
using phosphoramidite chemistry. It is precisely this point that
has caused confusion in the recent literature with reports
explicitely claiming that phosphoramidite chemistry would not
work in the presence of azide functionalities.14,32 On the basis
of our studies, we can confirm incompatibility only at the level
of nucleosides, as experienced by unsuccessful trials to
synthesize 2′-azido nucleoside phosphoramidite building
blocks; however, we and others observed clear compatibility
with strand elongation of azide containing DNA33,34 and
RNA.35 We further consider that the combination of
phosphotriester chemistry for azido nucleoside building blocks
and phosphoramidite chemistry for strand elongation could, in
principle, be replaced by a uniform phosphonate strategy (see
also ref 33). Nevertheless, we believe that a combined approach
(as used here) is highly attractive due to the widespread use of
phosphoramidite chemistry in most synthetic nucleic acid
laboratories, and therefore one may expect a more rapid
dissemination. We also mention that for RNA targets carrying
multiple azido groups a uniform phosphonate chemistry is
expected to be of higher efficiency due to straightforward
synthesis automatization.
With the synthetic tool in hands to generate 2′-azido
modified RNA at any of the four nucleosides, we were able to
study their structural features in detail. The crystal structure
revealed that the modification is very well tolerated and
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So far, the use of the Click reaction for RNA labeling has
relied predominantly on alkyne modified nucleic acids, while
the labeling partner was modified with an azide.39−45 The
feasibility of the present synthetic approach now opens reverse
attachment of the required functional groups, thereby
increasing flexibility. This will be of high significance for
selective and internal nucleic acid labeling with multiple dyes
on the same RNA, as for example requested by multicolor
single-molecule FRET techniques46 or for nucleic acid
cyclization,47 lariat formation or branching.48 The possibility
of reverse attachments will be of further interest in the
emerging field of RNA nanotechnologies.49,50
The 2′-azido modification accounts for the rare and
exceptional type of RNA modification that on the one hand
is well accommodated within the RNA, thereby leaving
biological recognition processes in cellular systems largely
unhindered, and on the other hand confers bioorthogonal
reactivity to the system. Allocating this functional group and its
properties at the ribose 2′ position that is equivalent in any of
the four canonical nucleosides makes this modification a highly
reliable and flexible tool for RNA chemical biologists.
METHODS
■
Figure 6. Internal labeling of selectively 2′-azido modified RNAs with a
fluorescent dye (F545) using Click chemistry. (A) Reaction scheme:
(a) 5 mM CuSO4, 10 mM sodium ascorbate, 50 °C, 3 h; cRNA = 1 mM,
cDye = 2 mM, H2O/CH3CN = 4/1; 60 μL total reaction volume. (B)
Reaction analysis of F545-alkyne and UGUCUUAUUGGCAGAGAC-
(CN319)TdG followed by anion exchange chromatography. (C)
Activities of G11-, C12-, and C19-labeled BASP1 siRNA and
corresponding controls (unmodified siRNA and siRNA with 3′-end
F545-labeled sense strand); for conditions see Figure 5. (D) BASP1
siRNA with internal (G11) 2′-azido-clicked F545 dye at the antisense
strand (bottom) and BASP1 control siRNA with standard 3′-end sense
strand labeling (top) show cytoplasmic localization in DF1 cells
visualized by fluorescence microscopy. The amounts of nucleofected
siRNAs were 0.24 nmol (C) or 0.48 nmol (D), respectively.
For the synthesis of 2′-azido building blocks 7 and 17 and for solid-
phase synthesis, deprotection, purification, and mass spectrometry of
2′-azido modified RNA, see the Supporting Information.
X-ray Crystallography. The 27-nucleotide SRL hairpin was
crystallized as described.25 This sequence was chosen as a test case
since crystallization conditions easily produce well-diffracting crystals.
Crystals were grown for 3 days at 20 °C for unmodified SRL sequence,
but 3 months were required for 2′-N3-A2670-modified SRL and 1 year
for 2′-N3-U2650-modified SRL. Crystals were cryoprotected for about
5 min in a reservoir solution containing 15% (v/v) of glycerol and 3.5
M ammonium sulfate and flash-frozen in liquid ethane for data
collection. The significant increase in time required for growing
crystals of both 2′-azido modified SRL sequences led to an important
non-merohedral crystal twinning that could be detected only during X-
ray diffraction (splitting of diffraction spots). Reasonably good data
could however be collected using the highly focused beam of the
X06SA beamline at the SLS synchrotron. Data were processed with
accommodated in the minor groove, with the formation of
water-bridged hydrogen bonding networks to the azide.
Compared to the structure of the unmodified RNA, the spine
of hydration is altered slightly, in a way that the larger azide
group replaces a water molecule. These minor structural
changes are in line with the hardly affected biophysical
characteristics obtained from CD and NMR spectroscopy.
Also, the influence on thermodynamic stabilites (analyzed by
UV melting curve analysis) were rather small.
To address the functional repertoire of 2′-azido modified
RNA, we explored two aspects, namely, siRNA applications and
bioconjugation. Concerning the first matter, the azide
modification accounts for a structurally non-perturbing
alteration, such as 2′-OCH3 and 2′-F, and like these behaves
in comparable manner with respect to its performance in the
siRNA system tested. In particular, the 2′-azido group possesses
the extraordinary property of being well accepted in the guide
(antisense) strand, while most other modifications are much
better tolerated in the passenger (sense) strand.8,31,36−38 The
2′-azido modification hence complements the existing set of
ribose modifications so far used in siRNA technologies,
however, with the major advantage that it brings in additional
chemical reactivity. This reactivity can be exploited for labeling
of the RNA as exemplified here by introduction of fluorescent
dyes.
the XDS Package.51 Structures were refined with PHENIX.52
significant twinning fraction (20.5%) was detected for 2′-N3-U2650-
modified SRL crystals. The structure was refined against twinned data
using PHENIX.
Click Labeling. For labeling, 2′-azido modified RNA (60 nmol)
was lyophilized in a 1 mL Eppendorf tube. Then, aqueous solutions of
F545 (Acetylene-Fluor 545, Click Chemistry Tools), CuSO4, and
sodium ascorbate were added consecutively; acetonitrile was added as
cosolvent30 to reach final concentrations of 1 mM RNA, 2 mM dye, 5
mM CuSO4, 10 mM sodium ascorbate, and a H2O/acetonitrile ratio of
4:1 in a total reaction volume of 60 μL. The reaction mixture was
degassed and stirred for 3−4 h under argon atmosphere at 50 °C. To
monitor the reaction and to purify the reaction mixtures, anion
exchange HPLC was used as described in the Supporting Information.
A
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
PDB codes 3DU8, 3S7C.
587
dx.doi.org/10.1021/cb200510k | ACS Chem. Biol. 2012, 7, 581−589

ACS Chemical Biology
Articles
selective conversion of ribonucleosides to 2′deoxynucleosides. J. Am.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ronald.micura@uibk.ac.at, e.ennifar@ibmc.u-strasbg.fr,
klaus.bister@uibk.ac.at.
ACKNOWLEDGMENTS
■
Funding by the Austrian Science Fund FWF (P21641 and I317
to R.M., P23652 to K.B.) and the Ministry of Science and
Research (GenAU III “Non-coding RNAs”; project P0726-012-
012 to R.M.) is acknowledged. We thank G. Hoffmann for
crystallization trials and V. Olieric for his support at the SLS
synchrotron.
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