© 2005 Nature Publishing Group
Silencing of microRNAs in vivo with ‘antagomirs’
Jan Kru ¨tzfeldt1, Nikolaus Rajewsky3, Ravi Braich4, Kallanthottathil G. Rajeev4, Thomas Tuschl2,
Muthiah Manoharan4& Markus Stoffel1
MicroRNAs (miRNAs) are an abundant class of non-coding RNAs
that are believed to be important in many biological processes
through regulation of gene expression1–3. The precise molecular
function of miRNAs in mammals is largely unknown and a better
understanding will require loss-of-function studies in vivo. Here
we show that a novel class of chemically engineered oligonucleo-
tides, termed ‘antagomirs’, are efficient and specific silencers of
endogenous miRNAs in mice. Intravenous administration of
antagomirs against miR-16, miR-122, miR-192 and miR-194
resulted in a marked reduction of corresponding miRNA levels
in liver, lung, kidney, heart, intestine, fat, skin, bone marrow,
muscle, ovaries and adrenals. The silencing of endogenous
miRNAs by this novel method is specific, efficient and long-
lasting. The biological significance of silencing miRNAs with the
use of antagomirs was studied for miR-122, an abundant liver-
specific miRNA. Gene expression and bioinformatic analysis of
messenger RNA from antagomir-treated animals revealed that the
in miR-122 recognition motifs, whereas downregulated genes are
depleted in these motifs. Analysis of the functional annotation
of downregulated genes specifically predicted that cholesterol
biosynthesis genes would be affected by miR-122, and plasma
cholesterol measurements showed reduced levels in antagomir-
122-treated mice.Ourfindings show thatantagomirs arepowerful
tools to silence specific miRNAs in vivo and may represent a
therapeutic strategy for silencing miRNAs in disease.
Approaches to the study of miRNA function in mammals have
focused on the overexpression or inhibition of miRNAs with anti-
as computational target predictions and validation by luciferase
reporter assays4–8. However, functional studies in mice that lack
specific miRNAs have yet to be reported. Further, because miRNAs
may be important in human disease6,9–12, approaches to interrupt
miRNA function may have therapeutic utility. Small interfering
double-stranded RNAs (siRNAs) engineered with certain ‘drug-
like’ properties such as chemical modifications for stability and
cholesterol conjugation for delivery have been shown to achieve
therapeutic silencing of an endogenous gene in vivo13. To develop a
chemically modified, cholesterol-conjugated single-stranded RNA
analogues complementary to miRNAs, and have termed these
To explore the potential of these synthetic RNA analogues to
silence endogenous miRNAs, we designed an antagomir, antagomir-
122, selective for miR-122, an abundant, liver-specific miRNA.
Antagomir-122 was synthesized starting from a hydroxyprolinol-
linked cholesterol solid support14and 2
This compound was administered to mice by intravenous injection
in a small volume (0.2ml) at normal pressure. Administration of
antagomir-122 resulted in a marked decrease in endogenous
miR-122 levels as detected by northern blots (Fig. 1a). Adminis-
tration of unmodified single-stranded RNA (anti-122) had no effect
on levels of hepatic miR-122 (Fig. 1a), whereas injection of uncon-
jugated, but chemically modified, single-stranded RNAs with a
Figure 1 | Specific targeting of miR-122 in mouse liver by tail-vein injection
of chemically modified single-stranded RNAs. a, Northern blots of total
RNA (15mg) isolated from mouse liver 24h after injection of differently
modified RNAs (three injections of 80mgkg21d21) against miR-122.
Samples were separated in 14% polyacrylamide gels in the absence (a, b) or
presence (c) of 20% formamide. Ethidium bromide staining of tRNA is
shown as a loading control. d, Mice were injected intravenously with PBS or
a miR-122–antagomir-122 duplex (three injections of 80mgkg21d21) and
the livers were harvested 24h after the last injection. Expression of miRNA-
122 was analysed by northern blotting.
1Laboratory of Metabolic Diseases, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.2Howard Hughes Medical Institute, Laboratory of RNA
Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.3Biology and Mathematics, Center for Comparative Functional Genomics,
Department of Biology, New York University, New York, New York 10003, USA.4Alnylam Pharmaceuticals Inc., 300 3rd Street, Cambridge, Massachusetts 02142, USA.
© 2005 Nature Publishing Group
partially modified (pS) or fully modified (fS) phosphorothioate
backbone and 2
122fS, respectively) led to an incomplete effect (Fig. 1a). The effects
of antagomir-122 were found to be specific because animals injected
with a control for antagomir-122 that harboured four mismatch
mutations (mm-antagomir-122) had no effect on miR-122 levels
(Fig. 1b). Furthermore, expression levels of let7 and miR-22 were
unaffected in mice treated with antagomir-122 and mm-antagomir-
122, indicating that silencing was miRNA-specific (Fig. 1b).
MicroRNA-122 is expressed at high levels in hepatocytes, with
miR-122 after treatment with antagomir was due to degradation or to
stoichiometric duplex formation between miR-122 and antagomir-
122, with the resulting inability of the probe to detect miR-122 in a
unconjugated anti-miR-122 oligonucleotides (anti-122fS and anti-
122pS) or antagomir-122 under stringent denaturing conditions
(Fig. 1c). No decrease in miR-122 levels could be detected with
unconjugated anti-miR-122 RNA-treated livers, showing that the
decrease in miR-122 levels observed under non-denaturing con-
ditions (Fig. 1a) was simply due to the formation of miR-122–RNA
duplexes. In contrast, miR-122 remained undetectable in livers of
mice treated with antagomir-122 and was not lost during the RNA
able to detect specific miR-122 degradation products when the
amount of miR-122 was increased by delivering preformed miR-
122–antagomir-122 duplexes into the liver (Fig. 1d). These data
indicate that the silencing of miRNA-122 in mice treated with
antagomir-122 might have been due to degradation of the
miRNA. The ability of antagomir-122 treatment to result in
miR-122 degradation is probably due to the efficient delivery
to hepatocytes and/or a consequence of changes in subcellular
localization of antagomir–miR-122 complexes.
Several pharmacological properties for antagomirs were evaluated
further, including dose–response, duration of action, and biodistri-
bution. To determine the dose of antagomir-122 that can completely
silence miR-122 we injected mice with a total dose of 80, 160 or
240mgper kg body weight and analysed for miR-122 expression
levels. The highest dose resulted in a complete loss of miR-122 signal
could be achieved after the injection of antagomir-122. Levels of
miR-122 were undetectable for as long as 23 days after injection,
indicating that silencing of miRNAs using antagomirs is long lasting
silencingactivityofantagomirs indifferenttissues, usingmiR-16asa
with antagomir-16, miR-16 was efficiently silenced in all tissues
tested except brain (Fig. 2a). Antagomir-16 did not seem to affect
the expression of the 89-nucleotide precursor of miR-16 detected in
bone marrow. The bioavailability of antagomir-16 was also assessed
by northern blotting in the above-mentioned tissue samples. Con-
sistent with the ability to silence miR-16 was our detection of
significant levels of antagomir-16 in all tissues except brain
(Fig. 2b). These data show that antagomirs achieve broad bio-
distribution and can efficiently silence miRNAs in most tissues
Many miRNA genes have been found to be located close together
result in long primary transcripts (pri-miRNAs) that are processed
by multiple enzymes in the nucleus and cytoplasm to generate the
mature miRNAs16. To investigate whether antagomirs targeting
polycistronic miRNAs retain their target specificity with no effect
on the expression of co-transcribed miRNAs, we injected mice with
antagomirs targeting either miR-192 or miR-194 of the bicistronic
cluster miR-192/194. Administration of antagomir-192 into mice
on the expression levels of miR-194. The converse effects were seen
0-OMe sugar modifications (anti-122pS and anti-
with injection of antagomir-194 (Supplementary Fig. 4). These data
show that antagomirs have the ability to differentially silence specific
miRNAs that derive from the same primary transcript.
MicroRNAs can regulate the mRNA levels of their targets17,18, and
regulated by miR-122 we performed gene-expression analysis with
mm-antagomir-122. In all, 363 transcripts were upregulated (at least
1.4-fold) and 305 transcripts were downregulated (at least 1.4-fold)
tary Table 1). The regulation of genes that were upregulated was
confirmed by reverse-transcriptase-mediated polymerase chain
reaction (RT–PCR; Fig. 3a). These included those members of gene
families that are usually repressed in hepatocytes, including those
Figure 2 | Antagomirs target microRNA expression in multiple tissues.
a, Northern blots of total RNA (10–30mg) isolated from different mouse
tissues 24h after injection of antagomir-16 (n ¼ 3). The precursor miRNA
was visible on northern blots of bone marrow. Membranes were probed for
miR-16. b, Total RNA from three mice as shown in a were pooled for the
detection of miR-16 and the injected antagomir-16. Ethidium bromide
staining of tRNA is shown as a loading control.
© 2005 Nature Publishing Group
encoding aldolase-A (aldo-A), N-Myc downstream regulated gene 3
(Ndrg3) and IQ-motif-containing GTPase-activating protein 1
(Iqgap1). Therefore, miR-122 could contribute to the maintenance
of the adult liver phenotype, as previously suggested for two other
upregulated and downregulated genes further, we analysed the 3
untranslated region (UTR) sequences of 9,554 mRNAs that have
cantly upregulated with at least a 1.4-fold change. Figure 3b shows
the percentage of genes that had at least one miR-122 recognition
motif CACTCC, corresponding to nucleotides 2–7 of miR-122—the
core ‘nucleus’ sequence19(also referred to as the ‘seed’ sequence20).
We observed a highly significant 2.6-fold increase in the probability
of having at least one miR-122 nucleus in the 3
0UTRs. Of these, 142 mRNAs were statistically signifi-
0UTR of upregulated
transcripts incomparisonwith genes with nochange inmRNAlevels
(Fig. 3b). Many of the miR-122 nucleus sequences in upregulated
genes had not previously been predicted19–21, indicating that the
number of direct miRNA targets might be significantly larger than
Forexperimental validation ofthelink betweenrepression andthe
presence of miR-122 nucleus matches within the 3
ing a miR-122 nucleus sequence into a luciferase reporter system.
When co-transfected with miR-122, all reporters exhibited signifi-
cant repression relative to co-transfections with control miRNA,
showing that miR-122 binding to its nucleus contributes directly to
mRNA repression (Fig. 3c). Of 108 transcripts that were significantly
downregulated, we observed that the probability of harbouring a
0UTR, we cloned
0UTR of five genes upregulated by antagomir-122 and contain-
Figure 3 | Positive and negative regulation of gene expression by
miRNA-122. a, Steady-state mRNA levels of genes in livers of mice treated
with mm-antagomir-122 or antagomir-122. Expression was measured by
in accordance with the International Standardized Nomenclature
(www.informatics.jax.org/mgihome/nomen/gene.shtml). Fold increase
indicates the ratio of expression levels of the means of mice treated with
antagomir-122 and mm-antagomir-122. The glyceraldehyde-3-phosphate
the miR-122 nucleus in differentially expressed genes. The plot shows the
percentage of genes with at least one miR-122 recognition motif present in
downregulated (upregulated) genes with a miR-122 nucleus was assessed by
1,000 random poolings of the same number of genes for each class from the
total setof transcripts. The resultfor downregulatedgeneswas significant at
0UTR. The significance of the lower (higher) percentage of
three standard deviations (P ¼ 0.001). The result for upregulated genes was
significant at more than eight standard deviations. c, Micro-RNA-directed
repression of Renilla luciferase reporter genes bearing 3
from genes identified from microarray expression analysis of antagomir-
122-treated mice after co-transfection into HEK-293 cells with the indicated
are shown as means ^ s.e.m., with n ¼ 6. d, For each of the 4,096 possible
hexamer RNA motifs and each transcript, the number of non-overlapping
occurrences divided by the length of the 3
motif, a non-parametric test (the one-tailed Wilcoxon rank sum test) was
applied to these distributions in upregulated versus no-change transcripts.
Shown is the histogram of the negative natural logarithm of all 4,096 P
values. e, Analogously to d, comparison between transcripts with no
significant change in expression and downregulated transcripts. Asterisk,
P , 0.05; two asterisks, P , 0.01; three asterisks P , 0.001.
0UTR was recorded. For each
© 2005 Nature Publishing Group
miR-122 nucleus was decreased by almost the same factor, 2.7-fold
(Fig. 3b). To analyse further whether the over-representation and
under-representation of miR-122 nucleus sequences are specific, we
analysed the abundance of all 4,096 possible hexamer motifs across
downregulated, upregulated and unchanged transcripts. When we
compared upregulated with unchanged genes, the miR-122 nucleus
sequence was the most significantly over-represented hexamer
(Fig. 3d). Notably, the miR-122 nucleus was within the top 1% of
under-represented motifs for downregulated transcripts (Fig. 3e),
indicating a possible evolutionary tendency of downregulated genes
to lack binding sites for miR-122. These results indicate that
upregulated mRNAs are directly targeted and repressed by miR-122,
but also that a significant number of downregulated genes are likely
to be activated by miR-122.
To assess the phenotype of altered gene regulation by miR-122, we
analysed the annotation of regulated genes for enrichment in Gene
Ontology categories. The top-ranking functional category was
‘cholesterol biosynthesis’ (P ¼ 1.6 £ 10211) and was found for
gene transcripts downregulated by antagomir-122. The expression
ofatleast 11genes involvedincholesterolbiosynthesis wasdecreased
between 1.4-fold and 2.3-fold in antagomir-122-treated mice (Sup-
plementary Table 1); some of these were confirmed by RT–PCR
(Fig. 4a). Mice injected with an adenovirus expressing miR-122
(Ad-122) increased the expression of some of these genes (Fig. 4b).
One of the gene transcripts downregulated by treatment with
antagomir-122 was 3-hydroxy-3-methylglutaryl-CoA-reductase
(Hmgcr), a rate-limiting enzyme of endogenous cholesterol biosyn-
thesis. We measured the enzymatic activityof Hmgcr in liverextracts
and found a roughly 45% decrease in activity in antagomir-122-
treated mice compared with mm-antagomir-122-treated mice
(9.7 ^ 1.0 and 17.2 ^ 2.3pmolpermg of microsomal protein per
minute, respectively; n ¼ 4, P ¼ 0.02). Consistent with this effect
was the observation that plasma cholesterol levels were decreased
more than 40% in treated animals whereas there was no detectable
effect on plasma non-esterified fatty acids, triglyceride, bile acid and
glucose levels (Fig. 4c). No decrease in cholesterol was observed with
antagomir-16, antagomir-192 and antagomir-194, showing that the
effects of antagomir-122 are sequence-specific and unrelated to the
use of a cholesterol-conjugated oligonucleotide itself. Decreased
cholesterol levels in antagomir-122-treated mice lasted for at least 2
weeks (data not shown). Antagomir-122 was well tolerated even
during the course of the prolonged treatment; no alterations in body
weight or serum markers of liver toxicity (alanine aminotransferase
(ALT) levels) were detected. Together, these data indicate that
miR-122 participates in regulation of the cholesterol biosynthetic
pathway and that silencing of a miRNA can be achieved without
The discovery of miRNAs is likely to change our understanding of
gene expression fundamentally, yet almost nothing is known of their
function in mammalian systems in vivo. Our data show that antago-
mirs can effectively silence miRNAs in vivo, and that antagomirs can
enable the study of gene regulation in vivo by a tissue-specific
miRNA, miR-122. In addition to many upregulated genes, silencing
of miR-122 also led to a decrease in a significant number of genes.
The mechanism by which miRNAs can activate gene expression is
remodelling) or an indirect effect (suppression of a transcriptional
repressor). Notwithstanding the large number of genes regulated by
antagomir treatment, it is striking, given the extent and duration of
miR-122 silencing, that the phenotype of antagomir-122-treated
mice was otherwise modest. Further studies are needed to explore
changes at the protein level as a result of silencing multiple miRNAs
as well as potential effects under stress conditions or in disease
models, but our data support the model that some miRNAs might
servemoreas a ‘rheostat’than as an ‘on–off switch’, to fine-tune gene
expression. Our novel pharmacological approach to silence miRNAs
specifically will allow the rapid generation of mice lacking specific
miRNAs or combinations of miRNAs for further functional studies.
Finally, because it has been shown that miRNAs are involved in
cancer9–12, cell growth and differentiation4,5,22, insulin secretion6and
a therapeutic strategy for diseases24such as cancer, hepatitis and
diabetes, and others almost certain to be discovered, in which
miRNAs participate in disease aetiology.
Figure 4 | MicroRNA-122 regulates the expression of genes involved in
cholesterol biosynthesis. a, RT–PCR analysis of cholesterol biosynthesis
genes identified in Affymetrix gene-expression analysis in livers of mice
the ratio of expression levels of the means of mice treated with mm-
antagomir-122 and antagomir-122. The glyceraldehyde-3-phosphate
dehydrogenase gene (Gapdh) was used as a loading control. Gene
abbreviations: Hmgcr, 3-hydroxy-3-methylglutaryl-coenzyme A reductase;
Dhcr7, 7-dehydrocholesterol reductase; Acas2, acetyl-coenzyme A
synthetase 2 (ADP forming); Mvk, mevalonate kinase; Hmgcs1, 3-hydroxy-
3-methylglutaryl-coenzyme A synthase 1; Fdps, farnesyl diphospate
synthetase; Sqle, squalene epoxidase; Fdft1, farnesyl diphosphate farnesyl
of cholesterol biosynthesis genes in animals 6 days after injection of Ad-
EGFP or Ad-122. The upper row shows a northern blot of liver RNA for
miR-122. c, Metabolic measurements in mice treated with antagomir-122
and mm-antagomir-122 control. FFA, non-esterified fatty acid. Asterisk,
P , 0.05; two asterisks, P , 0.01; three asterisks, P , 0.001.
© 2005 Nature Publishing Group
Synthesis of antagomirs. The single-stranded RNAs and modified RNA ana-
logues used in this study consisted of a 21–23-nucleotide length with modifi-
cations as specified: anti-122, 5
tides; subscript ‘s’ represents a phosphorothioate linkage; ‘Chol’ represents
cholesterol linked through a hydroxyprolinol linkage14; anti-122pS is anti-
miR-122 RNA with partial phosphorothioate backbone and anti-122fS is anti-
miR-122 RNAwith full phosphorothioate backbone. Details of the synthesis are
given in the Supplementary Methods.
Animals. All animal models were maintained in a C57BL/6J background on a
12-h light/dark cycle in a pathogen-free animal facility at Rockefeller University.
of saline or different RNAs (as indicated). RNAs were administered at doses of
in tissueswere performed24h after the lastinjectionunlessindicated otherwise.
Tissues were harvested, snap-frozen and stored at 2808C.
Generation of recombinant adenovirus. The recombinant adenovirus used to
express miR-122 (Ad-122) was generated by PCR, amplifying a 344-base-pair
miRNA precursor sequence with primers 5
The fragment was cloned into vector Ad5CMV-KnpA. Ad-EGFP (ViraQuest)
was used as a control. Mice were infected with 5 £ 109plaque-forming units per
mouse by injection into the tail vein.
was isolated three days after the initiation of treatment. RNA was pooled from
three animals for each group. The generation and analysis of Affymetrix
microarray data are described in the Supplementary Methods.
Northern blotting analysis. Total RNA was isolated with the Trizol reagent
(Invitrogen) and precipitation with ethanol. RNA was separated at 45mA on
14% polyacrylamide gels containing 8M urea and 20% formamide in a Protean
Nþnylon membranes (Amersham) in a Trans-Blot electrophoretic transfer cell
(3,000Cimmol21; NEN Life Science). Hybridizations were performed at 508C
NaCl, 75mM sodium citrate, 0.02% albumin, 0.02% polyvinylpyrrolidone,
0.02% Ficoll 400 and 0.1mgml21sonicated salmon-sperm DNA.
as described in the Supplementary Methods.
Assay of luciferase activity. Mouse full-length 3
amplified with specific primers and cloned downstream of the stop codon in
pRL-TK (Promega).HEK-293cellswere cultured in 24-well plates and eachwell
was transfected with 50ng of the respective pRL-TK 3
siRNA (Dharmacon). Cells were harvested and assayed 24–30h after transfec-
tion. Results were normalized to the Pp-luc control and are expressed relative to
the average value of the control miRNA (si-124).
A total of 17,264 3
obtained. Affymetrix probe identifiers were assigned to the Refseq transcripts
by using a mapping provided by Ensembl (http://www.ensembl.org/Multi/
martview). When more than one probe identifier mapped to a transcript, we
insisted that the Affymetrix significance call be consistent for all probes.
Transcripts were discarded otherwise. The fold change assigned to a transcript
was the average of all probes that mapped to the transcript. Finally, a cut-off of
0.5 in the log2of fold changes was applied.
Gene ontology analysis. The analysis is described in the Supplementary
Hmgcr activity assay. Hepatic microsomal Hmgcr activity was assayed by a
methodmodifiedfrom apreviously publishedprocedure anddescribedindetail
in the Supplementary Methods.
Statistical analysis. Results are given as means ^ s.e.m. Statistical analyses were
0; anti-122fS, 5
0; antagomir-122, 5
0; mm-antagomir-122, 5
0; antagomir-194, 5
0; antagomir-192, 5
0UTR sequences were PCR-
0UTR contructs (Rr-luc),
0UTR sequences and mapping of array probes to transcripts. We extracted
0UTR sequences at least 30 nucleotides in length were
performed with Student’s t-test, and the null hypothesis was rejected at the 0.05
Received 19 July; accepted 12 October 2005.
Published online 30 October 2005.
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Acknowledgements We thank J. Maraganore, V. Kotelianski and P. Sharp for
discussion and suggestions. These studies were supported by NIH grants (to
M.S., T.T. and N.R.), and an unrestricted grant from Bristol Myers Squibb (M.S.).
Author Information Gene expression data have been deposited at GEO-NCBI
under accession number GSE3425. Reprints and permissions information is
available at npg.nature.com/reprintsandpermissions. The authors declare
competing financial interests: details accompany the paper at www.nature.com/
nature. Correspondence and requests for materials should be addressed to M.S.