Synthesis of symmetrical thiol-adenosine conjugate and 5′ thiol-RNA preparation by efficient one-step transcription

Article abstract of DOI:10.1016/j.bmcl.2010.08.097

A symmetrical transcription initiator containing two adenosines and conjugated thiol functionality (ThioAMP dimer) is chemically synthesized. Transcription in the presence of ThioAMP dimer under the T7 Φ2.5 promoter yields 5′ thiol-labeled RNA (5′ HS-RNA) with up to 90% labeling efficiency, depending on the concentration ratio of ThioAMP dimer to ATP. The resulting 5′ HS-RNA may be used directly or after thiopropyl Separose 6B affinity column purification. Biotinylation of 5′ HS-RNA and formation of gold nanoparticle-RNA nanoplexes are demonstrated.

Full text of DOI:10.1016/j.bmcl.2010.08.097

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6254–6257
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of symmetrical thiol-adenosine conjugate and 50 thiol-RNA
preparation by efficient one-step transcription
⇑
Faqing Huang , Yongliang Shi
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406-5043, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A symmetrical transcription initiator containing two adenosines and conjugated thiol functionality (Thio-
AMP dimer) is chemically synthesized. Transcription in the presence of ThioAMP dimer under the T7
U
on the concentration ratio of ThioAMP dimer to ATP. The resulting 50 HS-RNA may be used directly or
after thiopropyl Separose 6B affinity column purification. Biotinylation of 50 HS-RNA and formation of
gold nanoparticle-RNA nanoplexes are demonstrated.
Received 26 July 2010
Accepted 19 August 2010
Available online 25 August 2010
2.5 promoter yields 50 thiol-labeled RNA (50 HS-RNA) with up to 90% labeling efficiency, depending
Keywords:
Transcription initiator
RNA labeling
Ó 2010 Elsevier Ltd. All rights reserved.
RNA biotinylation
Gold nanoparticle-RNA nanoplexes
Functionally derivatized RNAs have broad applications in chem-
istry and biomedical research.1–6 50 Thiol-labeled RNA (50 HS-RNA)
may be used to immobilized RNA on thiopropyl Separose 6B affin-
ity resins1 and on gold surfaces.7,8 Biotin-labeled RNA may be pre-
pared from 50 HS-RNA.1,4 Recently, 50 HS-RNAs have been used to
construct gold nanoparticle-siRNA (AuNP-siRNA) nanoplexes.9 In
addition, 50 thiol-functionalized RNA may be used to conjugate
with other macromolecules.10–15
transcription initiators that have been successfully used to label
the 50 end of RNA with high efficiencies.3,6,5,23 Herein we report
the synthesis of a symmetrical transcription initiator (4) that con-
tains two adenosines and a disulfide bond. Under transcription
conditions, the disulfide bond is reduced to a free thiol group.
Therefore, the resulting RNA transcript (5) is conjugated by a free
thiol group at the 50 end. Depending on the concentration ratio of
the transcription initiator and ATP, 50 thiol-labeling of RNA can
reach 90% labeling efficiency. The resulting thiol-labeled RNA
may be used directly in applications after a simple purification step
by membrane filtration. Additionally, thiol-labeled RNA may be en-
riched to near 100% purity by thiopropyl column purification.1 Fol-
lowing thiol-RNA preparation, we show that the RNA can be
further modified by biotin through the imidazole-catalyzed thioes-
terization reaction.4 Finally, we demonstrate the utility of our
thiol-labeled RNA for the preparation AuNP-RNA nanoplexes.
Synthesis of a symmetrical transcription initiator—ThioAMP dimer
(4). Starting with 6-aminohexanethiol (1, Dojindo Molecular Tech-
nologies), the transcription initiator ThioAMP dimer (4) was syn-
thesized in two chemical steps, as shown in Scheme 1. In the
first step, 1 was oxidized to form dithiobis(6-hexaneamine) (2)
by 0.5 molar equivalent of hydrogen peroxide oxidation in water,
pH 8. Extraction by dichloromethane followed by solvent removal
led to a white powder 2 (90% recovered yield), whose structure
was confirmed by NMR and MS: 1H NMR (400 MHz, DMSO-d6),
d = 1.248–1.647 (m, 20H, S–C–C4H8–C–NH2), 2.499 (t, 4H, J = 7.2
Hz, S–CH2), 2.691 (t, 4H, J = 7.2 Hz, N–CH2), MS calcd for
50 HS-RNA may be prepared by either chemical synthesis via
phosphoramidite chemistry,8,10,11,9,12–15 in vitro transcription,7,1–
3,16–21 or polynucleotide-catalyzed thiophosphorylation.8 For small
RNA sizes, 50 thiol-labeling of RNA by chemical synthesis is simple
and commercially available. However, RNA synthesis and 50 thiol-
labeling by phosphoramidite chemistry become impractical when
RNA sizes exceed a certain limit, typically around 60 nucleotides
(nt), due to low yields of full RNA sequences and high costs of
chemical synthesis. On the contrary, 50 thiol-labeling of RNA by
in vitro transcription is fast, relatively inexpensive for small quan-
tities, and unrestricted from RNA size limit. 50 Thiol-labeling of
RNA may be achieved by transcription under either the T7 class
II promoter (U2.5) or the class III promoter (U6.5). The former uses
a thiol-conjugated transcription initiator containing an adeno-
sine,1–3 while the latter utilizes thiol-containing guanosine deriva-
tives.7,16–21
We have previously demonstrated that transcription under the
T7
U
2.5 produces RNA transcripts that are superior to those pre-
pared under the T7
U
6.5.22 Taking the advantage of the T7
U
2.5
promoter, we have synthesized a number of adenosine-containing
C
12H28N2S2 [M+H]+ 265.18, found 265.17 (ESI).
In the second step, 2 was incubated at room temperature with
4 molar equivalent of adenosine 50-phosphorimidazolide (Im-
⇑
Corresponding author. Tel.: +1 601 266 4371; fax: +1 601 266 6075.
E-mail address: Faqing.Huang@usm.edu (F. Huang).
dAMP, 3)24 in DMF for 8 days to afford the symmetrical transcrip-
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.097

F. Huang, Y. Shi / Bioorg. Med. Chem. Lett. 20 (2010) 6254–6257
6255
H2O2
S
NH2
SH
H2N
S
H2N
2
Dithiobis(6-hexaneamine)
1 6-Aminohexanethiol
NH2
N
N
ImdAMP
3
O
P
N
(Adenosine 5'-
phosphorimidazolide)
N
N
N
O
O
O
OH
NH2
NH2
OH OH
N
N
N
N
N
N
O
H
N
O
N
S
N
P
O
O
P
O
N
H
S
OH
ThioAMP dimer
4
OH
OH OH
NH2
OH OH
Transcription DTT, NTP
φ2.5 promoter
NH2
N
N
N
O
S
N
N
N
O
N
HN
NH
N
O
NH Imidazole (Imd)
H
O
N
O
N
O
P
O
H
HS
N
P
O
O
OH
S
O
NH2
OH
OH
5
5' HS-RNA
N
N
7
5' Biotin-RNA
N
O
OH
HN
NH
O
O
RNA
N
O
O
P
RNA
O
OH
S
6
Biotin-AMP
HO
OH
Scheme 1. Chemical synthesis of transcription initiator ThioAMP dimer (4), enzymatic preparation of 50 thiol-labeled RNA (50 HS-RNA) by transcription, and synthesis of
biotin-labeled RNA (50 Biotin-RNA) by imidazole-catalyzed biotinylation.
tion initiator 4. After purification by HPLC (Fig. 1) and removal of
solvent by lyophilization, a white powder (ThioAMP dimer 4, 75%
recovered yield) was obtained. The following NMR and MS data
are consistent with the structure of 4: 1H NMR (400 MHz, D2O),
d = 0.937–1.275 (m, 16H, S–C–C4H8), 2.307 (t, 4H, J = 7.2 Hz,
S–CH2), 2.618–2.691 (m, 4H, S–C5–CH2), 4.019–4.049 (m, 4H, P–
O–CH2), 4.367 (dd, 2H, J = 1.6, 4.0 Hz, Hribose), 4.524 (t, 2H,
J = 4.4 Hz, Hribose), 4.782 (t, 2H, J = 5.2 Hz, Hribose), 6.102 (d, 2H,
J = 5.2 Hz, Hribose), 8.163 (s, 2H, HAr), 8.519 (s, 2H, HAr). MS calcd
for C32H52N12O12P2S2 [MꢀH]ꢀ 921.27, found 921.25 (ESI), calcd
for C32H52N12O12P2S2 [Mꢀ2H]2ꢀ 460.13, found 460.17 (ESI).
Preparation of 50 ThioRNA (50 HS-RNA, 5). Following our standard
transcription protocol,1,2,22,23 50-thiol-labeled RNA (50 HS-RNA, 5)
and in the presence of ThioAMP dimer 4. After transcription, RNA
transcripts and thiol-labeled RNA transcripts of relatively small
sizes (<100 nt) can be separated directly by polyacrylamide gel
electrophoresis (PAGE). Figure 2 shows RNA thiol-labeling under
different transcription conditions. The gel indicates that RNA
thiol-labeling yields can vary, depending on the concentration ratio
of ThioAMP dimer 4 and ATP. When the ratio is >8, RNA labeling
yields can reach 90%.
Synthesis of 50 Biotin-RNA (7): Biotinylation of thiol-labeled RNA
(50 HS-RNA, 5) may be achieved quantitatively in aqueous solutions
by reaction with biotinyl adenylate (Biotin-AMP, 6) in the presence
of imidazole.4 As can be seen from Figure 3, biotin-labeled RNA (50
Biotin-RNA, 7) can bind streptavidin to form stable biotin–RNA–
streptavidin complexes, which can be separated easily from RNA
by PAGE.1,4 The biotinylation yield of thiol-labeled RNA transcripts
(Fig. 3, lane 4) is the same as the RNA thiol-labeling yield (Fig. 2,
lane 3). After 50 HS-RNA was purified by thiolpropyl Sepharose
6B affinity column,4 RNA biotinylation yield reached 96%.
Preparation of gold nanoparticle-RNA (AuNP-RNA) nanoplexes: To
demonstrate the utility of 50 thiol-labeled RNA (50 HS-RNA) by the
above described transcription method, we prepared a 50 HS-RNA of
100 nt using a previous DNA template.5,23 After transcription, the
was prepared by in vitro transcription under the T7
U2.5 promoter
Figure 1. HPLC purification of ThioAMP dimer (4) and purify analysis of purified
ThioAMP dimer (inset). HPLC conditions were: Econosphere C18 (Alltech) 10 ꢁ
250 mm, flow rate 4 mL/min. The column was equilibrated with 20 mM phosphate
buffer (pH 7.0). Sample was loaded onto the column. At 18 min after sample
injection, the solvent was changed to 100% water. At 28 min, 10% MeOH was
introduced and the solvent was changed to 30% MeOH at 41 min. The major peak at
45.8–50 min (ThioAMP dimer) was collected. Its purity was confirmed (inset) by
analytical HPLC (Econosphere C18 (Alltech) 4.6 ꢁ 50 mm, flow rate 1 mL/min, 40:60
MeOH:20 mM phosphate buffer, pH 7.0).
Figure 2. Polyacrylamide gel electrophoresis (PAGE, 8%) analysis of 50 thiol-labeling
of RNA by transcription under different transcription conditions. In addition to ATP
and ThioAMP dimer 4,
a trace amount of [a-
32P] ATP was included in the
transcription solution to label RNA internally by 32P for the purpose of RNA analysis
by PAGE and phosphorimaging. Depending on the concentration ratio of ThioAMP
dimer and ATP, RNA 50 thiol-labeling can reach 90% efficiency. The RNA in the
experiment contained 35 nucleotides (nt).22 This was a high resolution (single
nucleotide resolution) gel, achieved by long gel-running time (1 h at 15 W for a
20 ꢁ 15 cm ꢁ 0.8 mm gel).

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F. Huang, Y. Shi / Bioorg. Med. Chem. Lett. 20 (2010) 6254–6257
Figure 3. PAGE (8%) analysis of 50 biotinylation of 50 HS-RNA (5) by Bioitn-AMP (6)
and imidazole. After reaction, unincorporated biotin was removed by EtOH
precipitation and the recovered RNA was incubated with streptavidin, followed
by PAGE. This was
a low resolution (not capable of resolving RNA at single
Figure 5. Formation of AuNP-siRNA nanoplexes from AuNPs and HS-siRNA
prepared by transcription and purified by thiopropyl Sepharose 6B affinity
column.1,4 The resulting RNA preparation contained near pure RNA (Fig. 3, lane
6). The same procedure as in Figure 4 was used to prepare AuNP-siRNA nanoplexes.
nucleotide resolution) gel, achieved by short gel-running time (10 min at 15 W for a
20 ꢁ 15 cm ꢁ 0.8 mm gel).1,4 The RNA in this experiment contained 100 nucleotides
(nt) that had been used in our previous studies.5,23
RNA sample was purified by Microcon (M30) filtration. The recov-
ered 50 HS-RNA was then used directly to prepare AuNP-RNA nano-
plexes following the Mirkin procedure.25 The final NaCl
concentration reached 0.7 M. The formation of AuNP-RNA nano-
plexes was monitored by measuringUV spectrum of AuNP solutions.
As can be seen from Figure 4, when AuNPs (13 nm diameter)26 were
mixed with transcriptionally-prepared 50 HS-RNA, the characteristic
520 nm plasma resonance peak of AuNPs red-shifted 9 nm. As a con-
trol, the same procedure was applied to AuNPs but without RNA.
AuNPs quickly formed aggregation and precipitated out of solutions.
The UV spectrum (Fig. 4) confirmed aggregation formation by NaCl.
As another control, AuNPs were mixed with unlabeled RNA of the
same sequence and size [prepared by transcription in the absence
of ThioAMP dimer (4)]. NaCl was then gradually added to the sample
to a final concentration of 0.7 M. As can be seen from Figure 4,
although there were some interactions between AuNPs and unla-
beled RNA, severe aggregation occurred and the dark red AuNP solu-
tion quickly turned to faint blue solution. With longer time, AuNPs
precipitated out of solution.
to prepare AuNP-siRNA nanoplexes following the same procedure
as described above. As indicated by UV spectra in Figure 5, our
transcriptionally-prepared HS-siRNA was able to form AuNP-siRNA
nanoplexes similar to those of AuNP-siRNA nanoplexes formed by
chemically synthesized 50 thiol-labeled siRNA.26 The UV spectrum
difference between the AuNP-RNA in Figure 4 and AuNP-siRNA in
Figure 5 are mostly like due to different RNA sizes (100 nt vs 27 nt).
In summary, we have synthesized a new symmetrical thiol-con-
taining transcription initiator (ThioAMP dimer 4). Transcription
under the T7
U2.5 promoter and in the presence of the ThioAMP
dimer produces 50 thiol-labeled RNA (50 HS-RNA) with up to 90%
labeling efficiency, depending on the concentration ratio of Thio-
AMP dimer and ATP. There is no sequence and/or RNA size limit
of the described RNA 50 thiol-labeling method. Although relatively
small RNAs can be conveniently prepared by phosphoramidite
chemistry, the described method is simple, highly efficient, and
relatively inexpensive. It is particularly useful for 50 thiol-labeling
of relatively large RNA molecules. For small RNA sizes, the method
is not only an option but also advantageous when the needed RNA
quantity is small. Finally, when a large number of different thiol-la-
beled RNA sequences are needed, this method is particularly
appealing.
To further demonstrate AuNPs and 50 thiol-labeled RNA interac-
tions, we prepared 50 thiol-labeled survivin siRNA27 and purified
HS-siRNA by thiopropyl Sepharose 6B affinity column according
to our standard protocol.1,4 The resulting HS-siRNA was then used
Acknowledgments
This work was supported by an NASA grant NNX07AI98G.
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