Pin-point chemical modification of RNA with diverse molecules through the functionality transfer reaction and the copper-catalyzed azide-alkyne cycloaddition reaction

Article abstract of DOI:10.1039/c1cc10582e

The internal modification of RNA has been successfully achieved by the functionality transfer reaction (FTR) and following click chemistry with diverse azide compounds. The benefits of the FTR have been demonstrated by its specificity, rapidity, broad applicability, and procedure simplicity.

Full text of DOI:10.1039/c1cc10582e

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COMMUNICATION
Pin-point chemical modification of RNA with diverse molecules through
the functionality transfer reaction and the copper-catalyzed azide–alkyne
cycloaddition reactionw
Kazumitsu Onizuka,ab Atsushi Shibata,ab Yosuke Taniguchiab and Shigeki Sasaki*ab
Received 29th January 2011, Accepted 4th March 2011
DOI: 10.1039/c1cc10582e
The internal modification of RNA has been successfully achieved
by the functionality transfer reaction (FTR) and following click
chemistry with diverse azide compounds. The benefits of the
FTR have been demonstrated by its specificity, rapidity, broad
applicability, and procedure simplicity.
the alkaline conditions or neutral conditions in the presence of
NiCl2 proceeds rapidly and selectively to lead to the modification
of the 2-amino group of the guanine base (Fig. 1B).8d
Knowledge of RNA is being rapidly enlarged such as seen in
the study of small RNA, in which a variety of functions of
miRNAs are being unrevealed in cell proliferation, cell death
and cancer progression, etc.1 Chemically modified nucleic
acids play a significant role in genomic research.2 Specifically
modified oligoribonucleotides (ORN) such as with a fluorescent
dye and a modified ribose skeleton can be obtained by
chemical synthesis.3 Non-natural nucleosides are randomly
incorporated into RNA by the biological processes.4 Site
specifically modified long RNA is obtained by ligation of
chemically modified short RNA to long RNA in the presence
of the template ODN by enzyme5 or photochemistry.6
A
Fig. 1 The functionality transfer reaction (FTR) for modification of
catalytic DNA with ligase activity was developed for the
internal modification of large RNAs.7 A simple method for
specific modification of RNA at the internal position without
the need of enzymes or a multistep procedure might be useful
for the study of function and structure of RNA. We have
recently developed the original approach based on the efficient
functionality-transfer reaction (FTR) within the DNA–RNA
duplex.8 The ODN probe incorporating 6-thioguanosine (GS)
is first functionalized with the 2-methyliden-1,3-diketone
transfer group. At the second step, the resulting functionality-
transfer ODN (FT-ODN) is hybridized with the target RNA,
and the functional group is selectively transferred to the
4-amino group of the cytosine base at pH 7 or the 2-amino
group of the guanine base under alkaline conditions (Fig. 1,
I to II). Site- and base specificities have been demonstrated using
non-complementary sequences.8b,c In particular, the FTR under
the 2-amino group of guanosine of the internal RNA.
These results encouraged us to establish a general method
for the internal modification of RNA with a variety of
molecules by using a copper(I)-catalyzed cycloaddition reaction
(‘‘Click’’ chemistry) with the acetylene-linked transfer group
and the azide compounds.9,10 We wish to report here that
Click-chemistry is applicable to the synthesis of the transfer
group for FTR and also the acetylene-modified RNA formed
by FTR serves as a scaffold for conjugation with a variety of
molecules (Fig. 1, II to III). The benefits of this system
have been demonstrated by its specificity, rapidity, broad
applicability, and procedure simplicity for the internal RNA
modification with varied molecules.
Fig. 2 gives an overview of the method for the internal RNA
modification through FTR and the ‘‘click chemistry’’ with a
variety of azide compounds. In one approach, the 1,3-diketone
transfer group having the acetylene unit of ODN1 (functional)
is transferred to the target RNA2 and then the resulting RNA2
(alkyne) serves as a scaffold for further modification by the
click chemistry with the diverse azide compounds.z
a Graduate School of Pharmaceutical Sciences, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
E-mail: saski@phar.kyushu-u.ac.jp; Fax: +81-92-642-6615;
Tel: +81-92-642-6615
b CREST, Japan Science and Technology Agency, 4-1-8 Motomachi,
Kawaguchi, Saitama 332-0012, Japan
In another approach, the diketone derivative 1 is first
transformed to the modified compound (2 or 3) by the click
chemistry, which is then introduced to the thio group of
6-thioguanosine of ODN1.
w Electronic supplementary information (ESI) available: Synthesis of
the compounds, and the experimental details for the functionality
transfer reaction and the Cu(I)-catalyzed cycloaddition reaction with
the RNA substrates. See DOI: 10.1039/c1cc10582e
c
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Fig. 2 Functionality transfer reaction for the internal RNA modification: the acetylene transferred to RNA works as a scaffold for labeling with a
variety of molecules through the ‘‘Click Chemistry’’.
The biotin (2) and the FAM linked diketone derivative (3)
were synthesized using 1 and the azide compound 8 and 9 in
the presence of CuSO4 and TBTA (Scheme 1). The functional-
ization of ODN1 was performed by using the diketone derivative
(1, 2 or 3) in the carbonate buffer at room temperature
at pH 10 for 10 minutes to produce the corresponding
ODN1 (functional) in more than 90% yield based on the
HPLC analysis (Fig. S2 and S3, ESIw). The resulting ODN1
(alkyne, biotin, FAM) was subjected to the functional group
transfer reaction with RNA2 in the carbonate buffer at pH 9.6.
The chemical yield of the modified RNA2 (alkyne, biotin,
FAM) was obtained by HPLC analysis (Fig. S2, ESIw). The
time courses of the reaction with ODN1 (alkyne) and ODN1
(biotin) are compared in Fig. 4. The reaction of ODN1
(alkyne) for rG was completed within 10 minutes, whereas
the reaction for the other bases was almost negligible
(Fig. 4A). The reaction of ODN1 (biotin long) and ODN1
(FAM short) proceeded similarly at slightly slower reaction
rates (Fig. 4B, and Fig. S4 (ESIw)).
We next investigated the second approach based on the click
chemistry between the alkyne-modified RNA2 (alkyne) and
Fig. 3 Representative azide compounds (R2–N3) used in this study.
the azide compounds shown in Fig. 3. ODN1 (alkyne) was
reacted with RNA2 (Y = rG) for 10 minutes as described
above, and the reaction mixture was used for the next click
chemistry by using the azide derivatives in the presence of
CuSO4. The FTR mixture was mixed with sodium ascorbate,
TBTA and the azide compound in DMSO–H2O solution, and
then the cycloaddition reaction was started by the addition of
Table 1 Chemical yield of the modified RNA2 by the click chemistrya
Yield (%)
10 min
50 min
Entry
Azide
Functional group
pH
1
2
3
4
5
6
7
8
4
5
6
7
8
9
9
10
11
12
12
PhCH2–
PEG
Pyrene
9.6
9.6
9.6
9.6
>99
>90
89
>99
90
32
83
69
31
69
—
>99
>99
—
>99
69
>99
>99
64
97
86
Biotin (short)
Biotin (long)
FAM (short)
FAM (short)
FAM (long)
Dabcyl (short)
Dabcyl (short)
Dabcyl (long)
9.6
9.6
7.3b
7.3b
9.6
9
10
11
7.3b
7.3b
65
a
The functionality transfer reaction was performed for 10 minutes,
then the azide compound (750 mM, DMSO), sodium ascorbate
(300 mM), TBTA (300 mM, DMSO), DMSO (DMSO/H2O = 3/7)
and CuSO4 (150 mM) were added to the mixture. The yield was
obtained by the HPLC analysis with monitoring UV at 254 nm. pH
was adjusted with 1% aqueous AcOH before the click reaction.
Scheme 1 Synthesis of the acetylene-attached diketone derivative (1),
its FAM, biotin derivative (2, 3), and the functionalized ODN1. (a) 8
(biotin long) or 9 (FAM short), CuSO4ꢀ5H2O, sodium ascorbate,
TBTA, DMSO/H2O = 9/1, rt, biotin: 48%, FAM: 90%. (b) ODN1
and 1, 2 or 3, in the carbonate buffer for 10 min, rt at pH 10, >90%.
b
c
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long linker produced lower reaction rates compared to those
with the shorter linker, probably because of their steric
hindrance. It should be also noted that the biotin group can
be easily attached to the internal site of RNA with the high
sequence and base selectivity. These results indicated the high
potential of the FTR for efficient and selective internal
modification of RNA. These results have demonstrated the highly
efficient labelling method for the internal position of RNA.
In summary, the internal RNA modification has been
successfully achieved by the functionality transfer reaction
(FTR) and following ‘‘click chemistry’’ with a variety of azide
compounds. The high specificity, rapidity, broad applicability
as well as simplicity of the procedure demonstrated in this
study emphasize the benefit of this system for the internal
modification of RNA. As the functionality transfer reaction
can be performed at neural pH in the presence of divalent
metal cations,8d the method described here is very unique and
is expected to be useful in the research of RNA.
Fig. 4 Time course of the FTR by using ODN1 (alkyne) (A), and
ODN1 (biotin) (B). A: 1.5 mM of ODN1 (alkyne), 1.0 mM of the target
RNA2(Y) in 50 mM carbonate buffer containing 100 mM NaCl at pH
9.6 at 25 1C. B: 15 mM of ODN1 and 10 mM RNA2(Y). The base
represents the base (Y) in the target RNA2(Y).
This work was supported by a Grant-in-Aid for Scientific
Research (S) from the Japan Society for Promotion of Science
(JSPS) and CREST from the Japan Science and Technology
Agency. We are grateful for the Research Fellowship from the
Japan Society for the Promotion of Science (JSPS) for Young
Scientists (K. O.)
Notes and references
z The nomenclature of the ODN and RNA is as follows: ODN1
(alkyne) represents the structure of ODN1 functionalized by 1, in
which the name in the parenthesis means the abbreviated name of the
substituent R1. RNA2 (alkyne) means that the guanine base of RNA2
is modified by the group with the substituent R1 = alkyne. The
products obtained by the Click chemistry are indicated as ODN1
(biotin short) or RNA2 (biotin short), in which the name in the
parenthesis means the abbreviated name of the group of R2 of the
azide compound.
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Fig. 5 The HPLC chart changes showing rapid and high-yield
modification of RNA by the Cu(I)-catalyzed Click chemistry.
(A) The reaction with PEG5000–N3 (5). (B) The reaction with
biotin (short)–N3 (7). All oligonucleotides were separated by
HPLC under the conditions described in the ESIw, and subjected to
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reaction are shown in Fig. 5. Surprisingly, the reaction with 4,
5, 6, 7 and 8 proceeded very rapidly; the reactions with 4 and 7
nearly completed in 2 min. The transformation yield was
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obtained by using all the azide compounds are summarized
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addition product rapidly in excellent yields (entries 1 and 4).
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3 and 5). FAM- and Dabcyl–N3 showed higher reactivity at
pH 7.3 than at pH 9.6. Probably, FAM is less negatively
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backbone of RNA. It is also likely that the Dabcyl group is
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to negatively charged RNA. The azide compounds with the
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c
5006 Chem. Commun., 2011, 47, 5004–5006
This journal is The Royal Society of Chemistry 2011