.
Angewandte
Communications
(TLR).[7] Conversely, some RBPs regulate pre-miRNAs by
interacting with their stem regions.[8,41,42] In an extension of
our work we labeled pre-let-7a-2 with biotin in its TLR
(ORN-27) such that immobilization on streptavidin-coated
beads would enable its stem region to capture RBPs. Cy3 was
added to position 62 of ORN-27 to confirm the immobiliza-
tion step. Interestingly, whereas the terminal Cy3 group of
ORN-31 prevented proper functioning of its 5p miRNA,
labels in internal positions (loop, 3p arm) of ORN-27 had no
such adverse effects on miRISC-mediated silencing in HeLa
cells. Thus, ORN-27 inhibited a let-7a luciferase reporter gene
similarly to unmodified pre-let-7a-2 (Figure 4c), showing that
it was processed correctly. Streptavidin-coated beads were
washed separately with solutions of ORN-27 and two mono-
labeled controls (ORN-13 and ORN-12 labeled with Cy3 and
biotin, respectively). Whereas beads washed with the controls
showed no Cy3 emission at 570 nm (not shown), ORN-27
gave a strong fluorescence (Figure 4d), suggesting it was
properly immobilized and that both biotin and Cy3 were
functioning correctly.
In summary, we have observed a poor reactivity of long
RNAs using published protocols for click conjugation reac-
tions. Through a careful experimental plan and HPLC-based
analysis, we have developed a reliable procedure for the
labeling of pre-miRNAs on solid support and we quickly
prepared a library of 26 mono- and bis-homo-labeled pre-
miRNAs in moderate to excellent conversion. Pre-miRNAs-
21, -106a, -122, -32, and let-7a-2 were all successfully
internally- and end-labeled with Cy3, BHQ-1, trioxalen, and
biotin indicating that the procedure is generally applicable.
Labeling efficiency was dependent on the position of the
modification in the sequence, and was lower close to the 3’-
end. Labeling on the 5’-end, in the 5p arm or the loop was
generally very efficient with conversion > 80%. Our con-
ditions were also compatible with the bis-hetero-labeling of
RNAs by resuming the automated synthesis after the first
click conjugation. Thus, four pre-miRNAs with combinations
of biotin/Cy3/BHQ were prepared in conversions of 20–61%.
Importantly, the use of regular 2’-O-TBDMS phosphorami-
dites and an easily prepared 2’-O-propargyl cytidine phos-
phoramidite makes the chemistry accessible to most oligonu-
cleotide synthesis laboratories. We have shown that this
approach is straightforward and broadly applicable and we
expect it to open new avenues in chemical biology of miRNA
precursors and other long non-coding RNAs of topical
interest.
Table 4: Hetero bis-labeling of pre-miRNAs.
Product
Pre-miRNA
Position of
the labels[a]
Labels
Conv.[b]
ORN-27
ORN-28
ORN-29
ORN-30
pre-let-7a-2
pre-miR-106a
pre-miR-122
pre-miR-32
loop/3p (31/62)
5p/3p (8/52)
5p/3p (11/52)
5p/3p (6/52)
biotin/Cy3
Cy3/BHQ
biotin/Cy3
BHQ/Cy3
20%
61%
41%
24%
[a] Nucleotide number of the modification from the 5’ terminus; 3p: 3p
arm; 5p: 5p arm. [b] Estimated conversion in the click step (calculated
from the peak integrals (A+B)/(A+B+C)).
It is often necessary to optimize the location of a label so
that the proper functioning of a pre-miRNA is not disturbed.
Pre-miRNAs are processed into mature miRNAs comprising
5p and 3p arms, either or both of which may be present and
active in cells.[39] Thus, synthetic pre-miR-122 produced active
mature miRNAs from both arms in HeLa cells: through its 5p
arm it inhibited dose-dependently the expression of a reporter
gene bearing a target site cloned from the GYS1 mRNA
(Figure 4a, left), and it suppressed a reporter bearing
a complementary target site for its 3p arm (Figure 4b, left).
Figure 4. Properties of single- and double-labeled pre-miRNAs. Pre-
miR-122 and ORN-31 were assayed for inhibition of luciferase report-
ers in HeLa cells (see Supporting Information): a) A reporter bearing
the GYS1 target site for the 5p arm of miR-122; b) a reporter bearing
a complementary target for miR-122-3p. c) ORN-27 and controls were
assayed against a let-7a-5p reporter. a–c) Error bars: single doses of 3
transfections. d) Empty magnetic streptavidin beads (i) and ORN-27
immobilized on magnetic streptavidin beads (ii: illustrated as cartoon)
(see Supporting Information) were imaged on an epifluorescence
microscope (AxioVision software). Two images were acquired: trans-
mission light and Cy3 fluorescence. The grayscale images were
processed with ImageJ (rsbweb.nih.gov/ij/) and merged. White scale
bars: 25 mm.
Pre-miR-122 labeled on its 5’-end with Cy3 (ORN-31:
Supporting Information) localized to the cytoplasm of cells
upon transfection (Figure S13). ORN-31 inhibited the 3p
target reporter (Figure 4b, right), but it showed little activity
against the GYS1 reporter (Figure 4a, right), strongly sug-
gesting that the 5’-label hindered the proper processing/
activity of its 5p miRNA, but not its 3p arm. Analogous results
were obtained with end-labeled pre-miR-32 (not shown). This
data is consistent with reports that alkylation of riboses close
to the 5’-end of siRNAs suppresses their silencing ability.[40]
Recently, we described the use of terminally-biotinylated
pre-miRNAs to capture from cells RNA-binding proteins
(RBPs) which interact with their terminal loop regions
Received: June 10, 2013
Revised: July 26, 2013
Published online: && &&, &&&&
Keywords: click reaction · phosphoramidites · pre microRNA ·
.
RNA hairpin · RNA labeling
[1] D. M. Hammond, A. Manetto, J. Gierlich, V. A. Azov, P. M.
[2] P. J. Hrdlicka, B. R. Babu, M. D. Sorensen, N. Harrit, J. Wengel,
4
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
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