Reversible 5′-end biotinylation and affinity purification of synthetic RNA

Article abstract of DOI:10.1016/j.tetlet.2004.09.019

Biotinylation using phosphoramidite A on a solid phase synthesizer gives biotinylated B and nonbiotinylated failure sequences; avidin-coated microsphere mediated affinity purification provides pure oligoribonucleotideC after treating with tetrabutylammonium fluoride. A reversible biotinylation phosphoramidite was synthesized and incorporated onto the 5′-end of an oligoribonucleotide on a solid phase synthesizer. After cleavage and deprotection, the crude synthetic oligomer mixture was incubated with NeutrAvidin coated microspheres, and the failure sequences removed by washing with a buffer followed by treating the microspheres with tetrabutylammonium fluoride to give a high quality unmodified full-length oligoribonucleotide.

Full text of DOI:10.1016/j.tetlet.2004.09.019

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7987–7990
Reversible 50-end biotinylation and affinity purification of
synthetic RNA
Shiyue Fang and Donald E. Bergstrom*
Department of Medicinal Chemistry and Molecular Pharmacology, 201 S University Street, Purdue University,
West Lafayette, IN 47907, USA
Walther Cancer Institute, Indianapolis, IN 46208, USA
Received 29 July 2004; revised 17 August 2004; accepted 2 September 2004
Available online 21September 2004
Abstract—A reversible biotinylation phosphoramidite was synthesized and incorporated onto the 50-end of an oligoribonucleotide
on a solid phase synthesizer. After cleavage and deprotection, the crude synthetic oligomer mixture was incubated with NeutrAvi-
dinꢀ coated microspheres, and the failure sequences removed by washing with a buffer followed by treating the microspheres with
tetrabutylammonium fluoride to give a high quality unmodified full-length oligoribonucleotide.
ꢁ 2004 Elsevier Ltd. All rights reserved.
Besides wide application in biology and medicine for
nonradioacitve labeling of biopolymers,1–3 the strong
taining a diisopropylsilyl acetal linkage, and their use for
reversible biotinylation, phosphorylation and affinity
purification of synthetic DNA.10,11 We demonstrated
that the phosphoramidites could be efficiently coupled
onto the 50-end of an oligodeoxyribonucleotide on a
solid phase synthesizer, and that the linkage was com-
pletely stable under synthetic and cleavage/
deprotection conditions. The biotinylated full-length
DNA could be efficiently attached to NeutrAvidinꢀ
coated microspheres, failure sequences could be com-
pletely removed by simple washing with buffers, and
high quality unmodified DNA could be recovered by
treating with fluoride ion. As it is well known, oligoribo-
nucleotide synthesis is generally less efficient than oligo-
deoxyribonucleotide synthesis, and thus, generates
more failure sequences. Moreover, extreme caution is
required to handle RNA because of ubiquitous RNAse.
These factors coupled with the higher hydrophilicity of
RNA make it more difficult to purify. Consequently,
an efficient, simple RNA purification method should
be valuable to the scientific community. Here we report
the synthesis of a reversible biotinylation ribonucleotide
phosphoramidite, its incorporation onto the 50-end of
an oligoribonucleotide on a solid phase synthesizer,
and the application of this biotinylation methodology
in affinity purification of synthetic RNA.
noncovalent
interaction
(association
constant
1015 Mꢀ1) between biotin and streptavidin or avidin
has been proved to be useful for efficient isolation of
biotinylated DNA from complex mixtures.4–6 Typically,
the mixture containing the biotinylated target is incu-
bated with streptavidin or avidin coated microspheres,
then, nonbiotinylated materials are removed by washing
with buffer followed by recovering purified target mole-
cules from the microspheres. In order to obtain unmodi-
fied DNA after affinity isolation, a special reversible
linker between biotin and target is required. The
reported linkers suitable for such a purpose include the
acid-cleavable triphenylmethyl alkyl ether linkage
reported by Gildea et al.4 and the photocleavable o-nitro-
benzyl alkyl phosphate diester linkage reported by
Rothschild and co-workers.5 Although successful in cer-
tain applications, these linkers have shortcomings such
as inconvenient preparation, long cleavage time under
acidic conditions and potential damage of biomolecules
caused by exposure to UV irradiation.7–9 Recently, we
reported the preparation of two phosphoramidites con-
Keywords: Oligoribonucleotide; RNA; Solid phase synthesis; Revers-
ible biotinylation; Phosphoramidite; Avidin; Affinity purification;
Diisopropylsilylacetal linker.
As shown in Scheme 1, the biotinylation phosphorami-
dite 1 was conveniently prepared from the known bio-
tinyl alcohol 210 in two steps. Thus, 2 (1equiv),
*
Corresponding author. Tel.: +1765 494 6275; fax: +1765 494
6275; e-mail: bergstrom@purdue.edu
0040-4039/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.09.019

7988
S. Fang, D. E. Bergstrom / Tetrahedron Letters 45 (2004) 7987–7990
1) Diisopropydichlorosilane,imidazole,
DIEA,DMF, 0oC,1h,rt,4h
O
H
N
S
O
OH
O
N
H
tBu
4 O
Imidazole,DMF,0oC,4h
O
2)
2
NH
N
NH
O
O
N
O
O
O
HO
O
O
O
OH
O
O
O
3
O
N
OMe
NH
P
O
N
N
H
N
5
O
S
O
O
O
O
Si O
O
N
O
tBu
4 O
H
O
O
Tetrazole,CH2Cl2,rt,12h
NH
OH
O
O
N
91%
4
O
O
O
O
NH
O
H
N
N
O
S
O
O
O
O
Si
O
O
N
tBu
O
4 O
H
O
O
NH
N
MeO
O
O
O
52%
1
P
N
O
O
O
Scheme 1. Synthesis of the reversible biotinylation phosphoramidite 1.
imidazole (1equiv) and diisopropylethylamine (DIEA,
3equiv) in dry DMF was cooled on an ice bath, diiso-
propyldichlorosilane was added, and the solution
was stirred at 0ꢂC for 1h and at room temperature for
4h. The resulting solution was added slowly to a solu-
tion of 20-O-ACE-uridine (3, 1equiv)12 and imidazole
(1equiv) in DMF via a cannula at 0 ꢂC, and the mixture
was stirred at the same temperature for 4h. Partition
between CH2Cl2 and 5% NaHCO3 and flash chromato-
graphy (SiO2, EtOAc/MeOH/Et3N = 95:4:1) gave 4 as a
white foam in 91% yield.13 The vacuum dried 4 (1equiv)
was dissolved in CH2Cl2 and degassed by argon fol-
lowed by the addition of phosphinylation agent 5
(1.3equiv) via a syringe, and tetrazole (1.1equiv) under
positive argon pressure. After stirring at room tempera-
ture for 12h, the mixture was partitioned between
CH2Cl2 and 5% NaHCO3. Purification by flash chroma-
tography (SiO2 pretreated with CHCl3/THF/
Et3N = 3:2:0.5, then washed with CHCl3/THF = 3:2;
after loading the sample, eluted with CHCl3/
THF = 3:2) gave product 1 as a white foam in 52%
yield.14
To test this biotinylation and affinity purification strat-
egy, the short 50-end biotinylated oligoribonucleotide 6
(Scheme 2) was next synthesized on a solid phase synthe-
sizer at Dharmacon, Inc. on a 1lmol scale.15,16 The syn-
thesis was carried out on a high cross-linked polystyrene
support using the 20-O-ACE protected phosphorami-
dites illustrated in Figure 1 and biotinylation phosphor-
amidite
1
as building blocks under standard
conditions. After complete deprotection and cleavage,15,16
O
NH
O
H
N
O
S
N
O
O
Si O
O
N
H
O
4 O
6
HN
NH
O
OH
O
P
O
O
GCCGAUUCACUGCU
O
O
5'
3'
TBAF,rt,12h
NH
N
O
HO
O
O
H
S
N
O
OH
O
N
+
O
OH
4 O
H
O
P
O
O
GCCGAUUCACUGCU
HN
NH
5'
3'
7
O
Scheme 2. Removal of biotin to generate unmodified RNA.

S. Fang, D. E. Bergstrom / Tetrahedron Letters 45 (2004) 7987–7990
7989
crude RNA (same amount loaded). As can be seen,
the biotinylated full-length RNA 6 runs much slower
than the unmodified full-length RNA 7 and failure
sequences, and the diisopropylsilylacetal linkage is not
completely stable under deprotection/cleavage condi-
tions. In order to estimate the degree of premature
breakage, a separate preparative denatured PAGE was
used to separate 6 and 7,20 and both were extracted from
the gel with NH4OAc buffer (0.1M). UV measurement
indicated that more than 70% 6 retained their biotin
moiety during cleavage and deprotection. Lane c is
recovered failure sequences removed from the Neutr-
Avidinꢀ gel (concentrated on SpeedVac, desalted on a
NAPTM-25 column, 1% loaded) during the affinity puri-
fication. This lane demonstrated the high efficiency of
the attachment of biotinyl RNA 6 to the avidin coated
microsphere as 6 is not visible. Lane d is the affinity
purified full-length unmodified RNA 7 (1% loaded),
and shows that the failure sequences have been com-
pletely removed yielding pure RNA.
4-N-Ac-C
U
B =
12
6-N-Bz-A
2-N-iPr-G
O
B
Si O Si
O
O
O
O
Si
O
O
MeO
O
O
O
P
N
O
O
Figure 1. Phosphoramidite monomers used for RNA synthesis.
the crude oligoribonucleotide (10% of the 1lmol synthe-
sis), which contains the full-length oligomer, failure
sequences, and other impurities, was incubated with
NeutrAvidinꢀ gel (600lL) in PBS buffer (300lL) at
room temperature for 2h.17 Because the failure
sequences generated in each synthetic cycle were capped
with large excess of acetic anhydride, only the full-length
oligoribonucleotide was biotinylated by the phosphor-
amidite 1. As a result, only the desired RNA was
attached to the microsphere through the noncovalent
interaction between biotin and NeutrAvidinꢀ, and the
failure sequences and other impurities were removed
by washing with PBS buffer (300lL · 3) and water
(500lL · 3). After drying on a SpeedVac, the pure
full-length unmodified RNA 7 (Scheme 2) was recovered
from the gel by treating with tetrabutylammonium fluo-
ride (TBAF, 1M, THF) at room temperature for 12h
with gentle shaking followed by quenching by equal vol-
ume Tris–HCl buffer (100mM, pH7.4), and washing
with water (300lL · 5). In order to estimate the recov-
ery yield of oligoribonucleotide from the microspheres,
in addition to the full-length sequence recovered from
gel, the failure sequences removed by washing were also
collected, both were desalted by a NAPTM-25 column,18
and their quantities were determined by UV measure-
ment at 260nm, respectively. From these data, the
recovery yield of RNA was estimated to be about 80%.
In summary, we prepared a novel reversible biotinyl-
ation phosphoramidite, and successfully incorporated
it onto the 50-end of oligoribonucleotide on a solid phase
synthesizer. To the best of our knowledge, this repre-
sents the first chemical biotinylation of the 50-end of
RNA using a reversible linker, which, upon cleavage,
affords unmodified RNA. We demonstrated that biotin-
ylated full-length RNA could be efficiently attached to
NeutrAvidinꢀ coated microspheres by simple incuba-
tion in a buffer, failure sequences, and other impurities
could be removed by simple washing, and high quality
RNA could be conveniently obtained by cleavage from
the microsphere.
Acknowledgements
Grant support from the National Institutes of Health
(GM53155) and the Showalter Trust Fund is gratefully
acknowledged. Assistance from the National Cancer
Institute Grant (P30 CA23168) awarded to Purdue Uni-
versity is also gratefully acknowledged.
The efficacy of the affinity purification was next evalu-
ated by 20% denatured (8M urea) polyacrylamide gel
electrophoresis (PAGE). In Figure 2, lane a is the crude
RNA treated with TBAF (1M, THF, 200 lL, N-methyl-
pyrolidinone, 200lL, rt, 12h; 10% of the 1lmol synthe-
sis treated, 1% of which loaded).19 Lane b is untreated
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.tetlet.2004.09.019.
References and notes
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Figure 2. PAGE analysis of the results of NeutrAvidinꢀ coated
microspheres mediated affinity purification of synthetic RNA. See text
for details.

7990
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1.22 (m, 12H), 1.25–1.28 (m, 6H), 1.32 (s, 9H), 1.47–1.87
(m, 8H), 2.05–2.06 (m, 6H), 2.18–2.31 (m, 4H), 3.05–3.08
(m, 2H), 3.22–3.49 (m, 10H), 3.50–3.70 (m, 8H), 3.72–3.98
(m, 15H), 4.32–4.43 (m, 1H), 5.21–5.25 (m, 1H), 5.48 (s,
0.5H), 5.56 (s, 0.5H), 5.67 (d, J = 8.1Hz, 1H), 5.99 (d,
J = 2.7Hz, 0.5 H), 6.02 (d, J = 3.9Hz, 0.5 H), 7.39 (d,
J = 8.4Hz, 2H), 7.58 (d, J = 8.4Hz, 2H), 7.85 (d, J =
7.5Hz, 1H); 13C NMR (CDCl3, 300MHz) d 10.06, 13.10,
13.67, 17.64, 17.75, 17.88, 20.85, 21.19, 24.64, 24.75, 25.37,
25.60, 29.80, 30.31, 31.15, 31.61, 34.22, 34.94, 35.51, 38.25,
39.08, 39.24, 40.05, 42.82, 42.92, 43.08, 45.46, 55.08, 57.37,
61.23, 61.48, 61.65, 61.88, 62.47, 62.68, 63.17,
67.93, 69.79, 69.86, 70.01, 70.09, 73.68, 83.46, 84.26,
87.85, 88.97, 101.89, 111.79, 111.96, 124.48, 125.50,
128.23, 128.97, 131.75, 135.80, 140.70, 140.94, 150.21,
150.30, 151.50, 154.88, 156.29, 163.58, 169.95, 170.87,
173.06, 173.33; 31P NMR (CDCl3, 300MHz) d 168.30,
168.57.
11. Fang, S.; Bergstrom, D. E. Bioconjugate Chem. 2003, 14,
80–85.
12. 20-O-ACE-uridine 3 was purchased from Dharmacon, Inc.
13. Biotinylurindine 4: Rf = 0.3 (EtOAc/MeOH = 9:1); IR
(thin film, cmꢀ1) m 3309 (br), 2945, 2864, 1735, 1685,
1653, 1247, 1111, 1051; 1H NMR (CDCl3, 300MHz) d
0.97–1.11 (m, 14H), 1.28 (s, 6H), 1.32 (s, 9H), 1.46–1.87
(m, 8H), 2.05 (s, 3H), 2.06 (s, 3H), 2.19–2.30 (m, 4H),
3.02–3.09 (m, 2H), 3.22–3.26 (m, 1H), 3.34–3.43 (m, 4H),
3.45–3.68 (m, 8H), 3.75–4.34 (m, 14H), 5.20–5.24 (m, 1H),
5.59, (s, 1H), 5.66 (d, J = 8.4Hz, 1H), 5.96 (d, J = 2.1Hz,
1H), 7.39 (d, J = 8.4Hz, 2H), 7.57 (d, J = 8.4Hz, 2H), 7.94
(d, J = 8.1Hz, 1H); 13C NMR (CDCl3, 300MHz) d 1.85,
13.26, 13.41, 17.69, 20.85, 25.40, 27.79, 27.95, 29.69, 29.78,
31.15, 31.66, 34.94, 35.62, 38.24, 39.09, 39.26, 40.01, 55.15,
57.44, 61.01, 62.49, 62.71, 62.79, 63.07, 68.16, 69.78, 70.04,
73.74, 84.17, 88.23, 101.97, 112.37, 116.52, 124.48, 128.97,
131.79, 140.15, 150.44, 154.91, 156.36, 163.67, 169.91,
170.92, 173.18, 173.61. MS (ESI, MꢀH) calcd for
C57H89N6O19SSi 1221.56, found 1221.35.
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Chem. Scand. 1990, 44, 639–641.
17. NeutrAvidinꢀ gel was purchased from Pierce Biotechno-
logy, Inc. PBS buffer: 136.9mM NaCl, 2.7mM KCl,
10.1mM Na2HPO4, 1.8mM KH2PO4, adjusted to pH7.0
with HCl.
18. NAPTM-25 column was purchased from Amersham Bio-
sciences. Desalting was performed following manufacture-
suggested procedure.
19. The gel was imaged on a Molecular Dynamics Storm (860)
system after staining in a SYBRꢀ Gold (purchased from
Molecular Probes, Inc.) solution in 1 · TBE buffer
(1:10,000) at rt for 30min.
20. RNAs 6 and 7 were identified with MALDI mass spectra.
Biotinyl RNA 6: calcd 5287, found 5289. Unmodified
RNA 7: calcd 4687, found 4687.
14. Phosphoramidite 1: Rf = 0.2 (CHCl3/THF = 1:1); 1H
NMR (CDCl3, 300MHz) d 0.97–1.12 (m, 14H), 1.12–