Small molecule inhibition of RISC loading.

Abstract

Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 μM), suramin (IC(50) = 0.69 μM), and oxidopamine HCL (IC(50) = 1.61 μM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.

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Small Molecule Inhibition of RISC Loading
Grace S. Tan,†,§Chun-Hao Chiu,‡,§Barry G. Garchow,†,§David Metzler,†Scott L. Diamond,‡,*
and Marianthi Kiriakidou†,*
†Department of Medicine and‡Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States
ABSTRACT: Argonaute proteins are the core components of the
microRNP/RISC. The biogenesis and function of microRNAs and endo-
and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA
binding and nuclease activities. Currently, there are no in vitro assays suitable
for large-scale screening of microRNP/RISC loading modulators. We
describe a novel in vitro assay that is based on fluorescence polarization of
TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we
identified potent small-molecule inhibitors of RISC loading, including
aurintricarboxylic acid (IC50= 0.47 μM), suramin (IC50= 0.69 μM), and
oxidopamine HCL (IC50= 1.61 μM). Small molecules identified by this biochemical screening assay also inhibited siRNA
loading to endogenous Ago2 in cultured cells.
R
The endogenous RNAi pathway intersects with the microRNA
(miRNA) machinery, which in mammals regulates gene
expression by inhibiting protein synthesis and inducing
mRNA decay.4,5Endo- and exo-siRNAs and miRNAs are
processed in the cytoplasm by the RNase III enzyme Dicer and
loaded to Ago proteins to form effector complexes (microRNP
or RISC). Endo-siRNAs modulate innate immunity in
plants,6−8Drosophila,9,10and C. elegans.11−13Although the
function of mammalian endo-siRNAs is not yet understood, it
is likely that they operate primarily in oocytes,14,15where
miRNA function appears to be non-essential.16In contrast to
the elusive function of mammalian endo-siRNAs, somatic
miRNAs in mammals regulate numerous biological processes,
and their disordered expression has been linked to human
diseases.17
In the past decade, RNAi directed by exogenously
administered small RNAs emerged as a powerful molecular
tool for gene-specific studies, complementing classic genetic
approaches. Like miRNAs and endo-siRNAs, exogenous
siRNAs are incorporated into Ago complexes.18Ago2 is an
RNA-guided endonuclease with the ability to form catalytically
active complexes with siRNAs/miRNAs and their precur-
sors.18−22In addition to its RNA binding and catalytic activity,
Ago2 competes with the cellular translation machinery23−25to
halt translation initiation. Ago2 has been implicated in the
biogenesis of siRNAs26and of miRNA subsets, either by Dicer-
independent processing of miRNA precursors27−29or by
mechanisms that are not yet clearly understood.30,31Due to
its role as regulator, carrier, and executor of miRNA/siRNA
function, Ago2 is at the core of the RNAi, and its regulation
directly affects the biology of miRNAs and siRNAs, at multiple
layers.
NA interference (RNAi) depends on double-stranded
RNA and induces sequence-specific gene silencing.1−3
Recently, novel approaches have emerged for the identi-
fication of chemical modulators of miRNAs. In this limited
number of recent studies, cell-based assays have been the
predominant methodology for large-scale screenings of
compounds that enhance or inhibit miRNAs or siRNAs.32−36
In general, these assays do not permit identification of RNAi
pathway components directly affected by the modulators. With
the exception of a recent report demonstrating inhibition of
Dicer’s function by small molecules,33in vitro assays utilizing
purified recombinant RISC factors have not been previously
reported.
In this study, we describe a novel method for large-scale
screening of chemical compounds that interfere with RISC
loading. In order to identify potential RISC modulators, we
used purified recombinant Ago2 to screen two collections of
small compounds: the Library of Pharmacologically Active
Compounds (LOPAC) and a custom collection of compounds
from the National Institute of Neurological Disorders and
Stroke (NINDS). Our studies established a novel in vitro
method that is based on fluorescence polarization (FP) of
TAMRA-labeled small RNAs and identified molecules that
inhibit RISC loading in vitro. Further testing using cell-based
assays demonstrated that compounds identified by large scale in
vitro screenings also inhibit de novo assembly of endogenous
RISC.
■RESULTS AND DISCUSSION
Small Molecule Inhibition of in Vitro RISC Recon-
stitution: Results of LOPAC and NINDS Compound
Libraries Screening. A total of 1,280 compounds from the
LOPAC library (final screening concentration 100 μM) and a
Received:
Accepted:
Published: October 25, 2011
July 20, 2011
October 25, 2011
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© 2011 American Chemical Society
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custom collection of 1,040 compounds (final screening
concentration 20 μM) from the National Institute of
Neurological Disorders and Stroke (NINDS) were screened
for potential inhibitors of miR-21 and Ago2 binding. The assay
is described in Methods and illustrated in Figure 1A. The
average Z-factor for the screening was ∼0.6, indicating a robust
assay.37A representative Z-factor plot is shown in Figure 1B.
Setting a 40% inhibition as a cutoff point, we detected 46 hits
from the LOPAC (hit rate 3.6%) and 21 hits from the NINDS
library (hit rate 2%). All hits were subjected to a 16-point, 2-
fold serial dilution (final concentration 50−0.0015 μM) dose−
response testing to determine the IC50 for Ago2:miR-21
binding inhibition. With confirmation dose−response testing of
the high-throughput screening (HTS) hits, a total of 17
compounds from the LOPAC and 8 compounds from the
NINDS library showed an IC50< 50 μM (confirmation rate of
37%).
DNA Binding Assays. To exclude nonspecific DNA-
binding inhibitors, we next performed a counter screen of the
candidate compounds that were confirmed in the dose−
response test. In this assay, compounds were tested for
competition of ethidium bromide (EtBr) binding to DNA
(average Z-factor of 0.81). Compounds with IC50< 50 μM in
the EtBr competition assay were then excluded because their
activity in the Ago2:miR21 FP assay was considered a result of
nonspecific nucleic acid binding. After filtering out compounds
that were DNA binders, 12 confirmed hits from the LOPAC
and 6 confirmed hits from the NINDS library remained. Three
compounds with the lowest IC50 values, PubChem SID
29221432 (compound 1, aurintricarboxylic acid (ATA), Figure
Figure 1. (A) Principle of the fluorescence polarization (FP) screening assay for RISC loading inhibition. TAMRA-labeled siRNA is free to rotate in
the absence of Ago2, resulting in a low polarization value. The large siRNA-loaded Ago2 complex rotates more slowly, resulting in a higher
polarization value. (B) Z-factor plot of one screening plate. Graphical representation of the results of one screening plate in FP HTS assay for RISC
loading inhibition. Active controls (◇) were located in wells 1−16 and 353−368 (columns 1 and 23). Neutral controls (□) were located in wells
17−32 and 369−384 (columns 2 and 24). Compounds (Δ) were tested in wells 33−352 (columns 3−22). With 3 standard deviation as a cutoff
point, 8 compounds were identified as hits (○) in this plate. The coefficient of variation (CV) of active and neutral control was 12.4% and 3.6%
respectively, with a Z-factor of 0.64. The assay protocol is described in Methods.
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2A), SID 29223713 (compound 2, oxidopamine hydrochloride
(HCL), Figure 2B), and SID 24277738 (compound 3, suramin
sodium salt, Figure 2C) were selected for cell-based assays. IC50
values were 0.47, 1.61, and 0.69 μM for ATA, oxidopamine
HCL, and suramin, respectively.
ATA Inhibits siRNA Function in Human Cells. The 3
miRNA inhibitors with the lowest IC50 values in the
biochemical screening assay were subsequently tested on the
function of endogenous RISC. Cells were co-transfected with
Renilla (RL) and Firefly (FL) Luciferase plasmids and then
treated with ATA, suramin, or oxidopamine HCL at a final
concentration of 25 μM. Subsequently, cells were transfected
with an siRNA targeting the coding sequence of RL (siLuc).
Normalized RL expression analysis revealed a robust de-
repression of RL by ATA and modest de-repression by suramin
sodium salt (Figure 3A). Oxidopamine HCL in the
concentration used had no effect on RL expression. Since
ATA was a potent inhibitor of siRNA activity in cell-based
assays, we next performed a dose−response experiment. Cells
were treated with ATA at concentrations ranging from 6.25 to
50 μM. Optimal de-repression of RL was achieved at
concentration of 25 μM. At higher concentrations (50 μM),
the efficiency of de-repression decreased, likely indicating a
saturation effect (Figure 3B). Next, we tested if ATA interferes
with siRNA inhibition of an endogenous gene using an siRNA
that targets RNA helicase A (RHA). We showed that in mouse
embryonic fibroblasts, ATA de-represses siRNA knockdown of
RHA (Figure 4). Together, these results indicate that ATA
inhibits RISC activity against transiently expressed reporters
and endogenous genes.
ATA Inhibits siRNA Loading to Endogenous Ago2.
We next examined the effect of ATA on the RNA binding and
nuclease activity of endogenous Ago2, in order to determine
whether ATA also inhibits RNA loading to Ago2 in cultured
cells. The RNA binding and catalytic activities of endogenous
Ago2 were tested in 293TRex cells treated with ATA or
oxidopamine HCL. The association of endogenous miRNAs to
Ago2 and the RISC activity of immunoprecipitated endogenous
Ago2 complexes were comparable in cells treated with ATA or
oxidopamine HCL (Figure 5A and B). Interestingly, while
endogenous miRNA:Ago2 complexes were not perturbed,
loading of exogenous siRNA (siLuc) to Ago2 was completely
inhibited by ATA (Figure 5A). These results are consistent with
those from our in vitro screening using recombinant Ago2 and
indicate that ATA inhibits RNA binding to endogenous Ago2
and de novo RISC assembly, without disturbing preformed
complexes of Ago2 with endogenous small RNAs or inhibiting
the catalytic activity of Ago2.
Recent studies have identified chemical modulators of
miRNAs and siRNAs32−36,38utilizing cell-based assays.
Figure 2. Structures and IC50curves of RISC loading inhibitors SID 29221432 (A), SID 29223713 (B), and SID 24277738 (C) found to inhibit
miR-21 loading to Ago2 in vitro.
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Enoxacin has been shown to promote the biogenesis of
miRNAs and to enhance their function.34In addition, inhibitors
and enhancers of miR-122, a miRNA required for hepatitis C
replication by the liver, have been recently reported.36In vitro,
Dicer processing of synthetic shRNAs modified to contain G-
quadruplexes is inhibited by porphyrazines and bisquinolinium
compounds.33In vitro assays utilizing purified, minimal RISC
have not been reported previously.
In our study, we undertook a novel approach by taking
advantage of our previously described in vitro RISC
reconstitution assay.21,22We established a robust in vitro
methodology to screen large chemical compound libraries for
modulators of RISC, by testing the binding of TAMRA-labeled
miR-21, a miRNA implicated in pathways controlling cell
proliferation and apoptosis,39to recombinant Ago2. Aurin-
tricarboxylic acid (ATA), oxidopamine HCL, and suramin
sodium salt reproducibly inhibited in vitro RISC reconstitution.
Suramin is a purinoceptor antagonist40with antihelminthic
and antiprotozoal activity.41It inhibits the activity of
deoxyribonucleic/ribonucleic acid (DNA/RNA) polymerase
and human immunodeficiency virus (HIV) reverse tran-
Figure 3. De-repression of siRNA-regulated Renilla Luciferase expression by ATA and suramin. (A) T-REx 293 cells were first co-transfected with
Renilla Luciferase (RL) and Firefly Luciferase (FL) plasmids. After treatment with ATA, oxidopamine-HCL, suramin sodium salt, or DMSO for 24
h, cells were transfected with Luciferase siRNA (siLuc). Results shown are average values of normalized RL/FL expression from 3 individual
experiments. (B) Dose−response curve of T-REx 293 cells co-transfected with RL and FL plasmids treated with varying amounts (DMSO/0−50
μM) of ATA, prior to siLuc transfection. The mean RL/FL ratio of 3 individual experiments is shown (error bars represent standard deviation).
Figure 4. ATA de-represses siRNA inhibition of endogenous RHA.
Mouse embryonic fibroblasts (MEFs) were treated with ATA or
DMSO and subsequently with RHA siRNA, as described in Methods.
RHA and β-tubulin were analyzed by Western blot.
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scriptase in vitro.42In our studies, suramin inhibited the binding
of miR-21 to Ago2 in vitro and had a modest effect in cell-based
assays.
Oxidopamine HCL is an adrenergic agent that has been used
extensively for the depletion of dopaminergic neurons and
experimental induction of Parkinson-like syndrome in
animals.43The molecular mechanisms of oxidopamine HCL
function are not well understood.44Although it potently
inhibited miR-21 loading to Ago2 in vitro, oxidopamine did not
interfere with loading of siRNA to endogenous RISC in cell-
based assays.
ATA is a triphenyl-methyl molecule that inhibits nucleases
and nucleic acid-binding enzymes.45,46When administered to
mice, ATA inhibits infectivity and replication of T cell viruses at
concentrations that are not toxic to the host cell.47It was
recently shown that ATA is a potent inhibitor of human flap
endonuclease inhibitor 1 (FEN1).48In vitro, as well as in cell-
based assays, ATA potently inhibited the loading of small RNAs
to Ago2. This inhibition was not sequence-specific, as indicated
by our experiments using three different RNAs (miR-21, siLuc,
and siRHA). Importantly, at the concentrations used in this
study, ATA blocked loading of siRNAs to endogenous Ago2
without affecting the nuclease activity of the protein or
dissociating preformed Ago2 microRNPs. This implies that
ATA targets RNA-free Ago2, either by inducing conformational
changes or, more likely, by directly blocking the RNA binding
domains of the protein. Preformed microRNPs are not affected,
likely because miRNAs bound to Ago2 preclude direct
interaction of ATA with the protein.
Our studies established a novel in vitro assay for high-
throughput screening (HTS) of chemical modulators of RISC.
This fluorescence polarization (FP) assay is simple, utilizes the
minimal RISC components, and can be readily used for large-
scale screenings. Since the assay allows for testing of individual
miRNA/siRNA loading to Ago2, it can be used to identify
modulators of specific miRNAs/siRNAs. RNAi is widely used
as a novel tool for studies of gene expression, regulation, and
function. To our knowledge, this is the first report of a tool that
can be used for high-throughput screening for modulators of
the RNAi executor. Furthermore, Ago proteins are also the
executors of the miRNA function. Thus our novel FP assay
provides a tool for HTS of modulators of a key protein in the
miRNA/siRNA pathway and can potentially have broad
applications in studies of the function of Ago and individual
miRNAs in cell biology and human disease.
Using our novel assay we identified ATA as a potent
inhibitor of de novo RISC assembly in vivo and in vitro, which
does not affect preformed endogenous miRNA complexes and
does not inhibit the catalytic activity of Ago2. Thus, ATA can
be used to study individual microRNPs or miRNAs, their half-
lives, and stability or to study the function of endogenous RNA-
free Ago2. Such studies should be planned taking into
consideration possible effects of the compounds on cell
biology. For example, ATA is a generic nuclease inhibitor,
with potentially deleterious effects at high concentrations.
However, in the relatively low concentration of 25 μM, ATA
selectively inhibits loading of siRNAs to Ago2 without affecting
other functions of the protein. Previous studies have shown that
ATA is associated with low toxicity in tissue cultures49and is
well-tolerated in vivo.50For in vitro studies of RISC assembly
using purified Ago2 and synthetic siRNAs, the direct effect of
ATA on siRNA loading versus potential “adverse” effect on the
nuclease activity of Ago2 can be assessed by different
experimental methodologies.
In summary, we established a tool for HTS of microRNP/
RISC modulators and identified compounds that can be used to
block the cycle of miRNA loading to Ago2, with applications in
RNAi and in studies of the biogenesis and function of miRNAs
in different tissues and cell lines.
■METHODS
Expression and Purification of GST-Ago2. Recombinant
mouse Ago2 was purified and tested for RISC activity as described
in ref 22.
High-Throughput Small Molecule Screening Assay. Volumes
of 1 μL of miRNA buffer (40 mM CH3COOK, 4 mM MgCl2, 1 mM
DTT), 0.5 μL RNasin Ribonuclease Inhibitor, and 2.5 μL RNase-free
water were dispensed into each well of 384-well assay plates. After
pintool transfer of 100 nL of compound from DMSO compound
plates (with pure DMSO in control columns) to the assay plate, a
volume of 2 μL of TAMRA labeled miR-21 and 4 μL of GST-Ago2
were added separately to each well at a final concentration of 3.75 nM
and 150 ng respectively, in the 10 mL final reaction volume. Columns
1 and 23 of each 384-well plate received 4 μL of water instead of GST-
Ago2 to provide the minimum polarization value for each plate.
Columns 2 and 24 received 100% DMSO instead of a chemical
compound in order to determine the highest polarization signal for full
binding of the fluorescent miR-21 by GST-Ago2. The means and
standard deviations of the “no-Ago2” control columns 1 and 23 and
the “DMSO” control columns 2 and 24 were used to calculate the Z-
factor for each plate to estimate the robustness of the assay (Figure
1B). The assay plates were incubated at RT for 30 min and
Figure 5. ATA inhibits de novo RISC loading. (A) T-REx 293 cells
were first co-transfected with RL and FL plasmids for 24 h, then
treated with ATA or oxidopamine-HCl or DMSO for 24 h, and then
transfected with siLuc. RNA was isolated from immunoprecipitated
Argonaute (Ago) complexes and analyzed by Northern blot. Probes
against siLuc and Let7-a were used as indicated. (B) In vitro RISC
assay with radiolabeled Let7-a target (TGLet7-a) perfectly comple-
mentary to Let7-a using endogenous immunoprecipitated Ago2
complexes. (C) Input T-REx 293 cell lysates used for Northern Blot
(A) and immunoprecipitation (B) analyzed for Ago2 expression by
Western blot.
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fluorescence polarization was measured (EnVision, PerkinElmer) to
calculate the millipolarization units (mP) [(mP = 1000(S − GP)/(S +
GP)]; mirror: BODIPY TMR FP Dual, excitation filter: BODIPY
TMR FP 531 nm, G = instrument factor, P = signal from PerkinElmer
BODIPY TMR FP P-pol 579 nm first emission filter, S = signal from
PerkinElmer BODIPY TMR FP S-pol 579 nm second emission filter).
A value of 3 × standard deviation was used to set the cutoff point to
select hits. Each hit from the primary HTS screen was then tested
twice using a 16-point, 2-fold dilution (final concentration 50−0.0015
μM) dose−response assay to determine the IC50for binding inhibition
of miR-21 to Ago2. Results were plotted and analyzed using an IDBS
XLfit model 205 to calculate IC50values.
DNA Binding Assay. Compounds were tested for their ability to
displace EtBr from DNA as an indication of the DNA-binding activity
of each compound. Vector pUC19 and ethidium bromide (EtBr) were
mixed to final concentrations of 8 μg/mL (pUC 19) and 5 μM (EtBr)
and dispensed to the intermediate plate for columns 2−22 and 24.
Columns 1 and 23 were the active control where the water and EtBr
mix was dispensed instead. Columns 2 and 24 were the neutral control
where 4 μL water was dispensed instead of chemical compound. For
dose−response tests, a volume of 100 nL of each compound from
DMSO stock plates was pintool transferred (final concentration 50−
0.0015 μM) to the assay plate (columns1, 2, 23, and 24 had 100%
DMSO instead of compounds). A volume of 6 μL of solution was
transferred from the intermediate plate to the assay plate and
incubated for 30 min at RT. The assay plate was read with EnVision
from PerkinElmer (mirror: general dual, excitation filter: 535 nM,
emission filter: 595 nM). The means and standard deviations of
neutral and active control were used to calculate Z-factor.
Source of Reagents Materials and Software Used for in Vitro
Screening. Sigma-Aldrich: Library of Pharmacologically Active
Compounds (LOPAC), potassium acetate (CH3COOK), magnesium
chloride (MgCl2), dithiothreitol (DTT). Promega: RNasin Ribonu-
clease Inhibitor.
Corning Incorporated: Corning 384-well low volume black round
bottom polystyrene NBS microplate. Greiner Bio-One North America
Inc.: polypropylene, 384 well, solid V-bottom storage microplate.
PerkinElmer Life and Analytical Sciences: EnVision Multilabel
Reader, Evolution P3 liquid handler. Thermo Scientific: Microdrop
384 Reagent Dispenser. ID business solution: IDBS XLfit.
Cell Culture, Transfection, and Luciferase Assays. T-REx 293
(Invitrogen) cells were cultured with Dulbecco’s Modified Eagle
Medium (DMEM) (Invitrogen) supplemented with 10% FBS (Sigma)
and L-glutamine (Mediatech). Cells were cultured in 100-mm dishes
and transfected at 40% confluency with 0.5 μg of pRLTK and 2 μg of
pGL3 plasmids, using Lipofectamine 2000 (Invitrogen). Twenty-four
hours after transfection, 3 × 105cells/mL were transferred to 6-well
plates and treated with 25 μM oxidopamine HCL (Sigma, H4381),
aurintricarboxylic acid (ATA) (Sigma, A1895), suramin sodium salt
(Sigma, S2671), or DMSO (Fisher) for additional 24 h. Treated cells
were then transfected with Silencer Renilla Luciferase siRNA
(AM4630) or Silencer Negative Control no. 1 siRNA (AM4611) in
Opti-MEM (Invitrogen) using Lipofectamine RNAiMax (Invitrogen).
Transfection media contained each of the above compounds at 25 μM
concentration. Twenty-four hours after siRNA transfection, cells were
harvested for Luciferase assays or immunopreciptation and Northern
blot. Luciferase assays were performed as described.51,52
For RHA RNAi, mouse embryonic fibroblasts (MEFs) were first
treated with ATA (25 μM) or DMSO for 24 h, as described above.
Treated mEFs were then transfected with of RHA siRNA (25 μM,
Dharmacon ON-TARGETplus SMARTpool DHX9 no. L-056729) for
an additional 48 h. Cells were harvested, and RHA protein was
detected by Western blot (RHA antibody: Abcam, ab26271, 1:1000).
β-Tubulin was used for loading normalization (E7 ascites:
Developmental Studies Hybridoma Bank, 1:2000).
RNA Immunoprecipitation, RISC Activity Assay, and West-
ern and Northern Blot. For RNA immunoprecipitation (RIP), cells
were lysed in buffer containing 20 mM Tris-HCl, pH 7.4, 200 mM
sodium chloride, 2.5 mM magnesium chloride, and 0.5% Triton X-100,
and endogenous miRNPs were immunoprecipitated as described in ref
51. Briefly, cell lysates were first mixed with 20 μL of protein G
agarose beads (Invitrogen) preincubated with 5 μL of monoclonal
anti-Ago antibody 2A853or 0.5 μL of nonimmune mouse serum, at 4
°C overnight. Agarose beads were then washed in lysis buffer. As
described previously,5110% of total protein G agarose volume was used
for Western blot, 10% for in vitro RISC assay, and 80% for Ago2-
bound RNA isolation and Northern blot. For Ago2 detection by
Western blot, 2A8 was used in 1:1000 dilution. In vitro RISC activity
assays were performed with endogenous immunoprecipitated RISC
complexes and a radiolabeled RNA target perfectly complementary to
endogenous Let-7a-3 (TGLet7-a), as previously described.22,54
Northern blots were hybridized with a 5′-end radiolabeled DNA
probe antisense to Renilla Luciferase siRNA (siLuc) at 37 °C
overnight or LNA probe antisense to Let7a at 42 °C overnight, and
hybridization signals were detected by autoradiography.
5′-End Labeling and Sequences of RNA, DNA, and LNA
Oligonucleotides. RNA, LNAand DNA oligonucleotides were 5′-
end radiolabeled with T4 polynucleotide kinase (NEB) as previously
described.51Oligonucleotide sequences:
TGLet7a: 5′-GUAUCAACCACUAUACAACCUACUACCU-
CAACGUUCAAUC-3′
Renilla Luciferase siRNA (siLuc) DNA antisense: 5′- TAG-
GAAACTTCTTGGCACC-3′
LNA-let-7a-3: 5′-AACTATACAACCTACTACCTCA-3′
■AUTHOR INFORMATION
Corresponding Author
*E-mail: kiriakim@uphs.upenn.edu; sld@seas.upenn.edu.
Author Contributions
§These authors contributed equally to this work.
■ACKNOWLEDGMENTS
This project was supported by the Transdisciplinary Awards
Program of the Institute for Translational Medicine and
Therapeutics (TAPITMAT) from the University of Pennsylva-
nia (M.K. and S.L.D.). The project was also supported in part
by Grant Number UL1RR024134 (S.L.D.) from the National
Center for Research Resources. The content of this report is
solely the responsibility of the authors and does not necessarily
represent the official views of the National Center for Research
Resources or the National Institutes of Health.
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