A click chemistry-based microRNA maturation assay optimized for high-throughput screening

Article abstract of DOI:10.1039/c6cc02894b

Catalytic enzyme-linked click-chemistry assays (cat-ELCCA) are an emerging class of biochemical assay. Herein we report on expanding the toolkit of cat-ELCCA to include the kinetically superior inverse-electron demand Diels-Alder (IEDDA) reaction. The result is a technology with improved sensitivity and reproducibility, enabling automated high-throughput screening.

Full text of DOI:10.1039/c6cc02894b

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DOI: 10.1039/C6CC02894B
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A Click Chemistry-Based microRNA Maturation Assay Optimized
for High-Throughput Screening†
Daniel A. Lorenza and Amanda L. Garner*ab
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Catalytic enzyme-linked click-chemistry assays (cat-ELCCA) are an inverse-electron demand Diels-Alder (IEDDA) chemistry has seen
increased usage due to its enhanced kinetics and lack of
requirement for a catalyst.7 In order to enable ready adaptation of
cat-ELCCA to generate reliable, high-throughput screening (HTS)-
compatible assays for any biological target, herein we describe our
optimization of the click chemistry-based detection step. Using our
miRNA maturation assay as a model (Fig. 2), we demonstrate that
cat-ELCCA can be employed utilizing IEDDA chemistry to yield an
assay with superior reproducibility and high-throughput potential.
emerging class of biochemical assay. Herein we report on
expanding the toolkit of cat-ELCCA to include the kinetically
superior inverse-electron demand Diels-Alder (IEDDA) reaction.
The result is
a technology with improved sensitivity and
reproducibility, enabling automated high-throughput screening.
Click chemistry is establishing itself as a critical tool in the
development and application of both in vitro and in vivo assays.1-3
Recently, we established a new assay technology, termed catalytic
Enzyme-Linked Click Chemistry Assay (cat-ELCCA), as a novel
approach for generating click chemistry-based assays armed with
catalytic signal amplification.3-5 In cat-ELCCA (Fig. 1), a biotinylated
biomolecule is first immobilized in the wells of a streptavidin-coated
microtiter plate. This substrate can already contain a click chemistry
handle or one can be added via an enzymatic reaction or
biomolecular interaction. Detection then occurs via an initial click
reaction with a labeled horseradish peroxidase (HRP), followed by
addition of a pro-absorbant, -fluorescent or -chemiluminescent HRP
substrate. Importantly, this catalytic system, similar to enzyme-
linked immunosorbent assays (ELISA), allows for increased
Click Chemistry
DNA/RNA/
protein
DNA/RNA/
protein
X
X
Y
HRP
Y
HRP
previous work:
N3
this work:
Me
Catalytic Signal Amplification
N
N
N
N
X
Y
=
H2O2
CuI-catalyzed
alkyne-azide cycloaddition
inverse-electron demand
Diels-Alder (IEDDA)
Substrate
Fluorescence
Absorbance
Chemiluminescence
Me
N
N
N
N
X
Y
=
N
HRP
HRP
Fig. 1 cat-ELCCA scheme with modular click chemistry detection.
sensitivity and
a significantly reduced risk of compound
microRNAs (miRNA) are important post-transcriptional
regulators estimated to regulate >60% of mRNA transcripts.8 These
small, ~22 nucleotide (nt) long RNAs are able to use their sequence
specificity to bind to the 3’ untranslated region (UTR) of
complementary transcripts and cause translational suppression.9
Due to their critical role in gene regulation, dysregulated miRNAs
have been implicated in many diseases, including cancer, diabetes,
and heart failure.9 To date, targeting miRNAs, and RNA generally,
interference from screening diverse libraries. Unlike ELISA,
however, it eliminates the requirement for enzyme-linked
secondary antibodies. To date, we have demonstrated this concept
for a post-translation modification3,
and microRNA (miRNA)
4
detection.5 Additionally, the first small molecule antagonist for
ghrelin O-acyltransferase (GOAT) was identified through a cat-
ELCCA-based screen of >4,000 compounds.4
Over the past 15 years, there has been an explosion in the
discovery and application of bioorthogonal reactions.6 While
copper-catalyzed azide-alkyne cycloadditions (CuAAC) remain the
most common, and the first generations of cat-ELCCA utilized this
reaction, some systems may be sensitive to copper or the
researcher may have a desire to use other chemistry. Recently,
11
with small molecules has remained a challenging goal.10, Using
cat-ELCCA, our goal is to discover new RNA-binding scaffolds that
can be used to inhibit pre-miRNA maturation, which is catalyzed by
Click
Chemistry
Dicer
X
No Signal
Streptavidin-coated
surface
Click
Chemistry
a.Program in Chemical Biology, University of Michigan, 210 Washtenaw Avenue,
Ann Arbor, Michigan 48109, USA
Dicer
X
X
Y
HRP
b.Department of Medicinal Chemistry, College of Pharmacy, University of Michigan,
428 Church Street, Ann Arbor, Michigan 48109, USA. Email: algarner@umich.edu
†Electronic Supplementary Information (ESI) available: General methods and
Inhibitor
Catalytic Signal Amplification
Fig. 2 cat-ELCCA for Dicer-mediated pre-miRNA maturation.
materials,
assay
protocols,
and
supplemental
figures.
See
DOI: 10.1039/x0xx00000x
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the RNase III enzyme Dicer that digests the precursor into a mature was observed only upon overexposure of the gel (FigV.ie3wbA, rbticolettOonmlin)e.
miRNA (Fig. 2),9 in a pre-miRNA-specific manner.
Although the rhodamine substrates reacted equally well with both
DOI: 10.1039/C6CC02894B
chemistries (qualitative due to overlap of the bands representing
pre-miR-21 and pre-miR-21-Rhod), coupling of RNA and protein was
significantly enhanced with the IEDDA reaction (reaction efficiencies
of 20% and 3% for IEDDA and CuAAC, respectively; see Fig. S1 of the
ESI). We attribute this difference to the 1,000-fold rate increase
reported with IEDDA over CuAAC and the absence of exogenous
catalyst,6,13 particularly since this system is untemplated.14-17 This
type of proximity effect is more likely to strongly affect CuAAC due
to its mechanism, which relies on formation of a metallacycle
involving a ligated CuI (one or two molecules), the azide and
alkyne.18, 19 Because of the presence of competing copper ligands
within biomolecules, this may explain why the rhodamine coupling
appears to be equivalent with CuAAC and IEDDA unlike the RNA-
HRP reaction. Finally, to verify that these modifications do not
interfere with Dicer-mediated pre-miRNA maturation, the TCO- and
mTet-labeled pre-miRNAs were subjected to Dicer cleavage.
Although more sterically bulky than the alkyne substitution used
previously,5 these modifications did not affect Dicer’s ability to
cleave the substrates (Fig. 3c).
Click
Chemistry
(a)
pre-miRNA
X
+
L
Y
pre-miRNA
X
Y
L
X
TCO
-
Y
L
mTet
mTet
mTet
TCO
TCO
TCO
Rhodamine
Rhodamine
HRP
1
2
3
4
5
6
7
TCO
mTet
-
Rhodamine
Rhodamine
HRP
mTet
N3
Rhodamine
-
N3
N3
7
Rhodamine
HRP
8
9
1
2
3
4
5
6
8
9
(b)
Rhodamine
Fluorescence
pre-miR-21-Rhod
pre-miR-21-HRP
(a)
(b)
SYBR GOLD
RNA Dectection
pre-miR-21
pre-miR-21-HRP
Overexposed
1
2
3
4
5
(c)
Fig. 4 IEDDA-based cat-ELCCA. (a) Comparison of TCO- and mTet-
labeled pre-miRNAs. (b) Comparison to CuAAC variant with respect
to raw chemiluminescence signal intensity in the absence of Dicer.
pre-miR-21
Dicer cleavage
products
25
Following confirmation of the individual steps of the assay, the
IEDDA-enabled pre-miRNA substrates were immobilized in the wells
of a 384-well streptavidin-coated plate. After immobilization, the
pre-miRNAs were subjected to Dicer cleavage, which removes the
chemical handle from the immobilized RNA (Fig. 2). The pre-miRNAs
were then reacted with the corresponding HRP conjugate.
Subsequently, the wells were washed, treated with SuperSignal
West Pico as a HRP substrate, and the resulting chemiluminescence
signal was measured. Importantly, regardless of which label was on
the RNA, there was a clear difference in signal intensity between no
enzyme and Dicer-treated wells (7.3- and 3.9-fold difference for the
TCO- and mTet-labeled pre-miRNAs, respectively) (Fig. 4a).
Interestingly, the signal intensity that we observed with the IEDDA-
based assay was much greater than that observed with the CuAAC
variant (25-fold enhancement; Fig. 4b).5 This is likely due to the
increased conversion of the IEDDA reaction, as observed by
fluorescence gel (Fig. 3b), and demonstrates increased sensitivity
with our optimized click detection step.
Since the pre-miRNA-TCO substrate yielded optimal signal-to-
background (S/B), we then further characterizated this second
generation cat-ELCCA for Dicer-mediated pre-miRNA maturation. As
Fig. 5ab shows, the assay is dependent on the concentrations of
Dicer and immobilized pre-miRNA. Moreover, it is general for other
pre-miRNA sequences (Fig. 5c). Finally, to examine how the assay
responds to the presence of a known RNA-binding molecule, we
tested DAPI (4’,6-diamidino-2-phenylindole; Fig. 5d,e), which acts as
an intercalator of RNA.20 High concentrations of DAPI (100 M)
were able to inhibit Dicer activity with statistical significance (p-
21
17
Fig. 3 Comparison of in-solution click reactions and Dicer cleavage.
(a) General scheme and table of click reactions. - = no pre-miRNA.
(b) pre-miRNA IEDDA products detected via fluorescence gel. Click
reactions were performed with 2.0 equiv. of L-Y for 2 h at 25 C.
Top = Detection of rhodaminepre-miRNA conjugate. Rhodamine
fluorescence was first detected, followed by addition of SYBR GOLD
for 5 min and RNA detection on the same gel. Middle = Detection of
HRPpre-miRNA conjugate using SYBR GOLD. Bottom
=
Overexposure and zoom of the band representing the HRPpre-
miRNA conjugate. (c) pre-miRNA maturation catalyzed by Dicer.
Lane 1 = miRNA ladder; Lane 2 = pre-miRNA-TCO; Lane 3 = pre-
miRNA-TCO + Dicer; Lane 4 = pre-miRNA-mTet + Dicer; Lane 5 = pre-
miRNA-mTet.
Our first step towards transitioning to IEDDA-based cat-ELCCA
was to determine the in-solution reactivity of our cat-ELCCA
substrates under typical assay conditions (Fig. 3a). We tested
conjugation of the highly reactive, albeit larger than our original
alkyne substituion,5 trans-cyclooctene (TCO) with mTet,12 and our
pre-miR-21 substrate (see the ESI for sequence) was labeled and
reacted with either a modified rhodamine or HRP (Fig. 3a). As
shown in Fig. 3b (lanes 16), successful coupling was observed with
both reactants. Importantly, when the pre-miRNA was reacted with
its paired HRP (lanes 3,6), a new band appeared indicating
formation of the RNA-protein conjugate. This is in stark contrast to
the results observed with CuAAC (lanes 79), where a weak band
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value <0.0001). Although this is likely through non-specific demonstrating the versatility of cat-ELCCA, we hope to promote the
interactions, it demonstrates that the assay can detect inhibition by application of this easily adaptable method to otVhieewr AbrtiicoleloOgniclinael
DOI: 10.1039/C6CC02894B
small molecules.
systems and targets. As cat-ELCCA is amenable to both CuAAC and
IEDDA click reactions, by combining these or other bioorthogonal
reactions, such as strain-promoted alkyne-azide cycloadditions, it
should be possible to follow multiple modifications allowing for
multiplexing of more complex systems. Future efforts beyond the
scope of this report will be dedicated to this goal.
(a)
(b)
This work was supported through a generous start-up package
from the University of Michigan College of Pharmacy, a pilot grant
from the University of Michigan Center for the Discovery of New
Medicines, and the NIH (R01 GM118329 to A.L.G. and P30
CA046592 to the University of Michigan Comprehensive Cancer
Center). D.A.L. is grateful for a fellowship from the Graduate
Assistance in Areas of National Need (GAANN) program. We thank
Martha Larsen and Steve Vander Roest for assistance with HTS
equipment set-up.
(c)
(d)
H2N
(e)
NH2
Notes and references
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Fig. 5 Characterization of IEDDA-based cat-ELCCA. Dependence on:
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DAPI binding. Significance was determined using a two-tailed,
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Fig. 6 HTS data with IEDDA-based cat-ELCCA.
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measured the Z’ factor, which is descriptive of dynamic range and
data variation associated with signal measurement (see Fig. S2 of
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excellent assay (Z’  0.5) (Fig. 6). This was coupled with excellent
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In conclusion, we have adapted cat-ELCCA to include IEDDA
reactions, providing enhanced signal intensity and a higher Z’ score
indicative of a more suitable high-throughput screening assay. By
This journal is © The Royal Society of Chemistry 20xx
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