Oligonucleotide-Addressed Covalent 3′-Terminal Derivatization of Small RNA Strands for Enrichment and Visualization

Article abstract of DOI:10.1002/anie.201701448

The HEN1 RNA 2′-O-methyltransferase plays important roles in the biogenesis of small non-coding RNAs in plants and proved a valuable tool for selective transfer of functional groups from cofactor analogues onto miRNA and siRNA duplexes in vitro. Herein, we demonstrate the versatile HEN1-mediated methylation and alkylation of small RNA strands in heteroduplexes with a range of complementary synthetic DNA oligonucleotides carrying user-defined moieties such as internal or 3′-terminal extensions or chemical reporter groups. The observed DNA-guided covalent functionalization of RNA broadens our understanding of the substrate specificity of HEN1 and paves the way for the development of novel chemo-enzymatic tools with potential applications in miRNomics, synthetic biology, and nanomedicine.

Full text of DOI:10.1002/anie.201701448

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201701448
German Edition: DOI: 10.1002/ange.201701448
RNA Recognition
Oligonucleotide-Addressed Covalent 3’-Terminal Derivatization of
Small RNA Strands for Enrichment and Visualization
+
+
ˇ
˙
Aleksandr Osipenko , Alexandra Plotnikova , Milda Nainyte, Viktoras Masevicius,
ˇ
Saulius Klimasauskas,* and Giedrius Vilkaitis*
Abstract: The HEN1 RNA 2’-O-methyltransferase plays
important roles in the biogenesis of small non-coding RNAs
in plants and proved a valuable tool for selective transfer of
functional groups from cofactor analogues onto miRNA and
siRNA duplexes in vitro. Herein, we demonstrate the versatile
HEN1-mediated methylation and alkylation of small RNA
strands in heteroduplexes with a range of complementary
synthetic DNA oligonucleotides carrying user-defined moieties
such as internal or 3’-terminal extensions or chemical reporter
groups. The observed DNA-guided covalent functionalization
of RNA broadens our understanding of the substrate specificity
of HEN1 and paves the way for the development of novel
chemo-enzymatic tools with potential applications in miR-
Nomics, synthetic biology, and nanomedicine.
systems, or modification of a specific position in a particular
tRNA.[10] However, none of these options is amenable for
addressable targeting of small RNAs. Recently, the HEN1
small-RNA methyltransferase from Arabidopsis thaliana[11–13]
has been exploited for selective covalent labeling of miRNA/
miRNA* duplexes for their enrichment and analysis.[14]
Naturally, HEN1 uses the cofactor S-adenosyl-l-methionine
(AdoMet) for the 2’-O-methylation of the 3’-terminal extend-
ing nucleotides in small-RNA duplexes but is inert towards
single RNA strands (Supporting Information, Figure S1). The
above strategy, methyltransferase-directed transfer of acti-
vated groups (mTAG), combined the cognate specificity of
the HEN1 methyltransferase and a synthetic AdoMet ana-
logue carrying a sulfonium-bound extended side chain instead
of the methyl group. However, practical implementation of
this approach has been limited owing to the short life-times of
mature microRNA duplexes in mammalian cells, which
S
mall non-coding RNAs (ncRNAs) such as microRNAs
(miRNAs) and small interfering RNAs (siRNAs) post-tran-
scriptionally control gene expression in eukaryotic organ-
isms.[1] They maintain genome stability and integrity, and
govern a wide range of human physiological and pathological
processes. Unique expression patterns of small RNAs for
different types of tissues, stem cells, disease stages, or therapy
responses have a great potential for diagnosis, prognosis, and
targeted development of the novel therapies for human
diseases.[2] Moreover, a high stability of ncRNA biomarkers in
biological fluids provides diverse opportunities for the devel-
opment of non-invasive monitoring methods.[3] Although
there is a range of general enzymatic and non-enzymatic
techniques for RNA modifications,[4–6] the exploration of non-
coding RNomes depends on reliable tools for selective
covalent labeling of small RNAs, which is difficult to achieve
using current methods.
contrasts with
a vast abundance of individual RNA
strands.[15,16] Given that DNA/RNA hybrids are known to
adopt a standard A-helix geometry characteristic of RNA
duplexes,[17,18] we assumed that non-methylatable oligonu-
cleotide probes could be used for hybridization with endog-
enous RNA strands to produce duplexes for selective
modification with HEN1. Thus, we examined several types
of model antisense oligonucleotides that bring certain prac-
tical advantages such as improved hybridization properties or
higher durability in cellular environments.
In accord with these expectations, we found that HEN1
was able to perform efficient 3’-terminal methylation of the
RNA strand in a series of model miRNA/DNA heterodu-
plexes (Supporting Information, Figure S2A and Scheme S1).
No HEN1-dependent modification was detectable in the
DNA strands, which lack the 2’-OH group but retain the
adjacent 3’-OH group in the 3’-terminal nucleotide, in DNA/
DNA or RNA/DNA duplexes (Figures S2B and S9A,
respectively), indicating a high regiospecificity of the reac-
tion. Remarkably, the rates of methyl group transfer onto
miR173 or miR173* strand under single-turnover conditions
in miR173/DNA173-R2:D2 and miR173*/DNA173-R2:D2
heteroduplexes were similar or even greater than those of the
natural miR173/miR173* substrates (Figure S2C and
Table S1). Typically, the methyl group transfer to a particular
RNA strand in a RNA/RNA duplex shows two kinetic
phases: A faster phase corresponding to direct binding and
modification of the target strand, and a slower phase
accounting for binding and release of the complementary
strand followed by rebinding and methylation of the target
strand.[11] In contrast, the methylation of miR173 and
miR173* strands in RNA/DNA heteroduplexes followed
The existing toolbox for targeted covalent labeling of
RNA includes methyltransferase-directed 5’-cap functionali-
zation in mRNAs,[7,8] internal programmable sequence-spe-
cific modification of RNAs using complex C/D box RNPs[9]
[*] A. Osipenko,[+] A. Plotnikova,[+] M. Nainyte, Dr. V. Masevicius,
ˇ
˙
ˇ
Prof. S. Klimasauskas, Dr. G. Vilkaitis
Institute of Biotechnology, Vilnius University
10257 Vilnius (Lithuania)
E-mail: saulius.klimasauskas@bti.vu.lt
giedrius.vilkaitis@bti.vu.lt
ˇ
˙
M. Nainyte, Dr. V. Masevicius
Faculty of Chemistry and Geosciences, Vilnius University
03225 Vilnius (Lithuania)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
https://doi.org/10.1002/anie.201701448.
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single-exponential kinetics. This may suggest that HEN1
poorly interacts with a DNA strand and thus effectively binds
the RNA/DNA duplex in a single orientation, positioning the
3’-terminal nucleotide of the RNA strand in the catalytic
center.
overhangs in double-stranded substrates for the HEN1
activity in vitro.[13,20] The broader substrate specificity of the
HEN1 methyltransferase observed in this study suggests that
the well-established preference for ncRNA duplexes with 3’-
dinucleotide overhangs in vivo may derive from interactions
with partner proteins such as HYL1 and/or DCL1 rather than
HEN1 itself.[21]
Crystallographic evidence[19] suggests that, in contrast to
the target strand, which is closely engulfed by the active site
and the so-called 5’-capping site, the 3’-terminus of the non-
target strand is exposed on the outer surface of the protein,
suggesting its tolerance to structural modifications or exten-
sions (Supporting Information, Figure S3A). Indeed, we
found no obvious effect on the methylation efficiency of the
RNA strand even with DNA probes that contained 3’-
terminal fluorophores (FAM or Cy5) or internal (Cy3) as
well as locked nucleic acid (LNA) derivatives (Figure S3B).
We then examined the modification of three miRNA-derived
(miR173, miR-26a, and let-7a) strands in RNA/DNA hetero-
duplexes bearing 0–7 nucleotide 3’-overhangs on the DNA
strand and found that all of the examined substrates could be
faithfully methylated by HEN1 under standard experimental
conditions (Supporting Information, Figures S4 and S5, top
row). Moreover, efficient methyltransferase activity in vitro
was observed with blunt-ended RNA/DNA as well as RNA/
RNA duplexes that contained no 3’-overhangs on either end
(Supporting Information, Figure S6). Slight variations of the
single-turnover methylation rates observed with the blunt-
ended and 3’-overhang RNA/RNA duplexes (Table S1) argue
against a previously suggested requirement of dinucleotide 3’-
We next determined whether these RNA/DNA hetero-
duplexes can be selectively modified by HEN1 using synthetic
AdoMet cofactor analogues carrying extended side-chains
with functional amino (2–3), azide (4), alkyne (5), or biotin
groups (6–7) (Figure 1) as previously reported for RNA/RNA
duplexes.[14] Indeed, we found that the extended side-chains
were efficiently transferred to the RNA strand in a range of
RNA/DNA duplexes (Figure 1 and the Supporting Informa-
tion, Figures S7 and S8); the modification levels with
cofactors 2–6 were in the range of 70–98%. ESI-MS analysis
(Supporting Information, Figure S9 and Table S2) of the
nucleoside composition in HEN1-modified RNA/DNA
hybrids confirmed the transfer of a full-length side chain on
the ribose moiety of a 3’-terminal nucleotide. The nature of
the 3’-terminal ribonucleotide had a minor influence on the
HEN1-directed alkylation efficiency (C, 96–99%; G, 92–
96%; A, 76–92%; U, 62%) (Supporting Information, Fig-
ure S10), suggesting that the presented method is suitable for
labeling a diverse variety of types and isoforms of small
ncRNAs. Moreover, the methyltransferase tolerated 3’-ter-
minal and internal DNA modifications such as covalently
Figure 1. Selective oligonucleotide-directed 3’-terminal 2’-O-labeling of short RNA strands using HEN1 methyltransferase. A) RNA of interest is
annealed to a complementary oligodeoxynucleotide (i), optionally containing 3’-terminal (ii) or internal (iii) affinity or fluorescence reporters,
multiple modifications (iv) or a 3’-terminal DNA aptamer (v) followed by HEN1-catalyzed transfer of extended side chains containing functional or
reporter groups from synthetic cofactor analogues 1–7 for two-step (compounds 1–5) or one-step (compounds 6–7) 2’-O-labeling at their 3’-
terminal nucleotides. B) Gel-electrophoretic analysis of representative examples of 3’-terminal modification of miRNAs with the Ado-11-amine
cofactor 3 in heteroduplex structures (i–v) depicted on the left; solid arrows point at bands corresponding to covalently modified RNA strands,
dotted arrows to input RNAs. C) Synthesis of Ado-13-biotin 6 from Ado-6-azide 4.
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attached amino groups, FAM, or Cy3 fluorophores as well as
structure interacted much more efficiently with the magnetic
an oligonucleotide containing several internal locked nucleic
acid (LNA) nucleotides (Figure 1).
beads than SA210-R2:s0 alone (Supporting Information,
Figure S16) suggesting that hybridization with the target
RNA enhanced the accessibility of the aptamer by eliminat-
ing fold-back interactions in the DNA strand.
To demonstrate that a synthetic DNA probe can selec-
tively address the HEN1-mediated labeling to a predefined
miRNA strand, we conducted the alkylation reaction in
a mixture of three distinct miRNAs. The obtained data clearly
showed that only the miRNA strand that was complementary
to the added DNA probe was efficiently modified (Support-
ing Information, Figure S11A). We further explored the
selectivity of the method in multicomponent RNA mixtures.
As shown in Figure S11B, the addition of up to a 100-fold
excess of E. coli total RNA did not alter the modification
efficiency of the spiked miR173 or miR-210 strands. More-
over, both a standard and a 3’-FAM-modifed DNA strand
efficiently directed the derivatization of miR173 in the
presence of a 10-fold excess of total RNA from U2OS
osteosarcoma cells (Figure S11C).
In real biological systems, the DNA-directed HEN1-labeling
of target RNA is prone to producing detectable background
signal owing to labeling of native miRNA/siRNA duplexes. This
shortcoming could be resolved by using two-reporter detection
whereby one reporter group (fluorophore, quencher, or affinity
tag) is synthetically incorporated into the targeting DNA probe
and the other reporter is selectively deposited in the mTAG
reaction (Supporting Information, Figure S17). As an example,
we tested whether our aptamer-based approach is suitable for
selective click-labeling and subsequent affinity extraction of the
desired ncRNA from a complex RNA pool. For this, we
prepared four miR-26a, miR173, miR-210, and let-7a mixtures
in which only one of the miRNAs was 5’-P33-labeled and thus
visible on PAGE. After annealing with the miR-210-specific
DNA aptamer, the samples were incubated with HEN1 and
Ado-6-azide cofactor followed by labeling with a Cy5.5-alkyne
dye. Only miR-210 was selectively labeled (Supporting Infor-
mation, Figure S18) and effectively enriched using streptavidin
beads (Figure 2). The fluorescent band of the magnetic beads-
bound (Bd) fraction co-localized precisely with the position of
the P33-miR-210 band, confirming that the Cy5.5 fluorophore
was selectively attached to miR-210 (Figure 2 and Figure S18,
bottom panels).
In the second step, a range of commercially available
reporter groups such as fluorophores or biotin can be
conjugated to the covalently derivatized RNA strands to
afford its selective visualization or purification (Supporting
Information, Figures S12B–D and S13). Alternatively, a one-
step biotin labeling of miRNA can be achieved using
appropriate extended cofactor analogues (Figure S12A). As
the modification yields of some miRNAs using the previously
described Ado-18-biotin cofactor[14] proved rather low, we
prepared a new enantiomerically pure analogue, Ado-13-
biotin 6, from the above-mentioned azide cofactor 4 (Fig-
ure 1C). A high yield conjugation of 3-biotinoylamidoprop-1-
yne was achieved by copper-assisted alkyne–azide cycloaddi-
tion (click) reaction under mildly acidic conditions. The new
cofactor exhibited a significantly higher (87% versus 19%)
trans-alkylation activity towards the least favorable miR-210/
DNA210-R2:D2 heteroduplex (Supporting Information, Fig-
ure S14). Akin to the AdoMet-dependent methylations
described above, HEN1-directed transalkylations showed no
obligatory requirement for the 3’-overhangs of the target
RNA strand (Figure S8). Moreover, in many cases we found
that shifting the DNA sequence to form blunt-ended hetero-
duplexes with target RNAs led to a higher efficiency of RNA
modification. An even higher tolerance has been observed
towards variations of the 3’-overhangs of the DNA strand
(Figure S5, bottom row). Finally, we examined if HEN1 could
tolerate a large functional ligand at the 3’-terminus of the
guide oligonucleotide, which could be suitable for the
development of novel biotechnological or therapeutic appli-
cations. As a proof of principle, DNA oligonucleotides
complementary to miR173 or miR-210 were extended at
their 3’-termini to include a 29-mer streptavidin-specific DNA
aptamer St-2-1.[22] Using these substrates, we observed that
HEN1 was able to both methylate and alkylate the miRNA
strands in duplexes regardless of their 3’-overhang structure
(Supporting Information, Figure S15). Insertion of a tetranu-
cleotide spacer between the aptamer and the complementary
DNA sequences further improved the modification efficiency.
The alkylated miRNA/aptamer was compatible with purifi-
cation using streptavidin-coated magnetic beads. Importantly,
we found that the double-stranded miR-210/SA210-R2:s0
A proof of principle experiment of a two-fluorophore
assay utilizing Fçrster resonance energy transfer (FRET) in
Figure 2. Aptamer-addressed selective labeling and extraction of
miRNA. Four mixtures of miR-26a, miR173, miR-210, and let-7a
miRNAs each containing different P33-labeled miRNA were annealed to
a streptavidin-specific aptamer SA210-R2:j0 complementary to miR-
210. Samples were incubated with HEN1 and Ado-6-azide 4 cofactor,
click-labeled with Cy5.5 alkyne fluorophore and immobilized on strep-
tavidin-coated magnetic beads. P33-miRNAs were analyzed in input
(In), supernatant (Sn), and magnetic beads (Bd) fractions after
separation on a denaturing polyacrylamide gel. Cy5.5 fluorescence was
detected mainly in (In) and (Bd) but not in (Sn) fractions.
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Acknowledgements
ˇ
˙
˙
˙
The authors thank Audrone Ruksenaite and Georgij Kostiuk
for valuable help with MS and FRET analysis. This work was
supported by the Research Council of Lithuania (grant
number MIP-059/2015 to G.V.) and the NIH (HG007200 to
S.K.).
Conflict of interest
The authors declare no conflict of interest.
Keywords: HEN1 · methyltransferases · microRNA · siRNA ·
synthetic AdoMet analogues
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microRNA strand through HEN1-directed covalent alkyny-
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Altogether, we demonstrate a novel chemo-enzymatic
tool for addressable 3’-terminal covalent functionalization
and labeling of individual small-RNA strands in vitro. This
paves the way to a broad range of potential applications of
this approach in miRNomics research, nanomedicine, and
next generation analytical tools.
Manuscript received: February 9, 2017
Revised: March 27, 2017
Final Article published: && &&, &&&&
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These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
RNA Recognition
Versatile RNA tagging: A method for
addressable chemo-enzymatic labeling of
miRNAs and siRNAs, which exploits the
activity of HEN1 2’-O-methyltransferase
to transfer functional groups to the 3’-end
of the RNA strand in DNA/RNA hetero-
duplexes, is presented. This method
permits a variety of user-defined single-
or dual-reporter applications, in which
one tag can be synthetically incorporated
into the DNA probe and the second is
covalently attached by HEN1 to the target
RNA.
˙
A. Osipenko, A. Plotnikova, M. Nainyte,
ˇ
ˇ
V. Masevicius, S. Klimasauskas,*
G. Vilkaitis*
&&&& — &&&&
Oligonucleotide-Addressed Covalent 3’-
Terminal Derivatization of Small RNA
Strands for Enrichment and Visualization
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!