Obad et al Supp Info - Nature · PDF filecocktails (P2850, P5726, P8340, Sigma) and lysed by passing three times through a 21-gauge needle and another three passes through a 25-gauge

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SUPPLEMENTARY INFORMATION

Silencing of microRNA families by seed-targeting tiny LNAs

Susanna Obad1, Camila O. dos Santos2, Andreas Petri1, Markus Heidenblad1, Oliver Broom1, Cristian Ruse2, Cexiong Fu2, Morten Lindow1, Jan Stenvang1, Ellen Marie Straarup1, Henrik Frydenlund Hansen1, Troels Koch1, Darryl Pappin2, Gregory J.

Hannon2 and Sakari Kauppinen1,3

1Santaris Pharma, Kogle Allé 6, DK-2970 Hørsholm, Denmark

2Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA

3Copenhagen Institute of Technology, Aalborg University, Lautrupvang 15, DK-2750 Ballerup, Denmark

TABLE OF CONTENTS SUPPLEMENTARY NOTES .................................................................................................... 2

Cell culture .............................................................................................................................. 2 Western blot analysis .............................................................................................................. 2 Detecting off-target effects using Sylamer .............................................................................. 2 Proteomics ............................................................................................................................... 3

SUPPLEMENTARY FIGURES ................................................................................................ 5 Supplementary Figure 1. Inhibition of miR-21 function by tiny seed-targeting LNA. ............ 5 Supplementary Figure 2. Assessment of specificity and optimal length of LNA-antimiRs targeting the miR-21 seed region. ........................................................................................... 6 Supplementary Figure 3. Subcellular localization of antimiR-21 in HEK293 cells. .............. 7 Supplementary Figure 4. Specificity of tiny antimiR-122 and antimiR-155. .......................... 7 Supplementary Figure 5. Inhibition of miR-17, miR-18 and miR-19 families by 8-mer tiny LNAs. ....................................................................................................................................... 8 Supplementary Figure 6. Silencing of the let-7 miRNA family by seed-targeting tiny LNA in PC3 cells. ................................................................................................................................ 9 Supplementary Figure 7. Uptake of 35S-labeled antimiR-21 in mouse tissues over time after systemic administration of a single intravenous dose of 10 mg/kg. ...................................... 10 Supplementary Figure 8. Targeting of miR-21 in a mouse breast tumor model by tiny antimiR-21. ............................................................................................................................ 11 Supplementary Figure 9. Assessment of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice. ................................................................. 12 Supplementary Figure 10. The impact of phosphorothioate backbone modifications on in vivo silencing of miR-122 in the mouse liver by tiny seed-targeting LNA. ........................... 13 Supplementary Figure 11. Effect of tiny LNA mediated inhibition of miR-21 and let-7 in vitro. ...................................................................................................................................... 14 Supplementary Figure 12. Effect of tiny LNA mediated silencing of let-7 and miR-122 on the mouse liver transcriptome. .................................................................................................... 15 Supplementary Figure 13. Proteomic analysis of PC3 cells transfected with tiny antilet-7. 16 Supplementary Figure 14. Inhibition of (a) let-7 and (b) miR-155 function by 8-mer tiny LNAs. ..................................................................................................................................... 16

SUPPLEMENTARY TABLES ................................................................................................ 17 Supplementary Table 1. AntimiR oligonucleotide sequences. ............................................... 17 Supplementary Table 2. Repetitive human genes removed prior to Sylamer analysis. ........ 18 Supplementary Table 3. Repetitive mouse genes removed prior to Sylamer analysis. ......... 18 Supplementary Table 4. Oligonucleotides used to generate miRNA luciferase reporters. ... 19 Supplementary Table 5. List of PCR primers used in this study. .......................................... 19

REFERENCE LIST .................................................................................................................. 20

Nature Genetics: doi.10.1038/ng.786

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SUPPLEMENTARY NOTES

Cell culture PC3 cells (ECACC #90112714) were cultured in DMEM medium (Sigma), supplemented with 10% fetal bovine serum (Biochrom AG) and 25 ug/ml Gentamicin (Sigma). HeLa cells (ECACC #93021013) were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1x NEAA and 25 ug/ml Gentamicin. Huh-7 cells (a gift from R. Bartenschlager, Dept Mol Virology, University of Heidelberg) were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 1x NEAA and 25 ug/ml Gentamicin. HepG2 cells (ECACC #85011430) were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin. RAW264.7 cells were grown in DMEM supplemented with 10% FBS, 4mM Glutamax I and 25 µg/ml Gentamicin. Lipopolysaccharide (LPS) was purchased from Sigma and stimulation of RAW264.7 cells was carried out by treating cells with 100 ng/ml LPS. 4T1 cells (ATCC #CRL-2539) and Phoenix-Ecotropic producer cell line (G. Nolan, Stanford University, Stanford, CA) were cultured in DMEM medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and Penicillin/Streptomycin (Invitrogen). HEK293 cells stably expressing the FLAG-tagged Ago2 protein (a gift from H. Soifer, Montefiore Medical Center, Albert Einstein College of Medicine) were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 0,2 mg/ml Gentamicin.

Western blot analysis The affinity purified rabbit serum antibody Pdcd4 (Rockland), the mouse monoclonal BTG2 antibody (Abcam), the rabbit monoclonal PU.1 antibody (Cell Signaling), the monoclonal mouse antibody p27 (BD Biosciences), rabbit polyclonal anti-HMGA2 Peptide 1 (Oxis), the rabbit monoclonal RAS antibody (Cell Signaling), goat polyclonal Aldolase A (Santa Cruz), monoclonal anti-tubulin alpha (Ab-2) (Neomarker), the monoclonal anti eIF2C2 (Abnova) and the mouse monoclonal GAPDH antibody (Abcam) were all used according to the manufacturers’ instructions.

Detecting off-target effects using Sylamer We used the Sylamer1 method to produce the landscape plots in Supplementary Figs. 11 and 12. Sylamer1 is an algorithm that analyzes ordered lists of genes to find over- or underrepresented sequence ‘words’ of pre-defined lengths. We ordered the gene list according to the t-statistic for difference between LNA-antimiR treated and control samples. The landscape plot shows significance of the occurrence biases for sequence words across the gene ranking. Functional evidence for miRNA inhibition can be seen as an overrepresentation of the corresponding seed match site among the up-regulated mRNAs. We would expect potential off-target effects arising from direct binding of an oligonucleotide to a binding site in a mRNA to result in a bias of words complementary to the sequence of the oligonucleotide. To investigate if treatment with tiny LNA-antimiRs causes such effects, we supplied the algorithm with sequence information on the entire transcripts (the longest spliced transcript for each gene in the ENSEMBL database). Initially, this resulted in identification of clusters of significant words forming very narrow peaks at the extreme ends of the some of the ranked gene lists (not shown). However, detailed examination of these peaks showed that they originated from few highly repetitive genes that contained up to 50 repeats of various


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8-nucleotide words (Supplementary Tables 2 and 3). Except for Prg4 in the mouse having a single complementary match to the 8-mer antimiR-122 sequence, none of the genes described in Supplementary Tables 2 and 3 have complementary matches to any of the antimiRs or LNA scramble control used in these studies. In order not to obscure the Sylamer analyses with these “repeat artefacts”, we have excluded the genes listed in Supplementary Tables 2 and 3. Supplementary Figs. 11 and 12 show the resulting 6-, 7-, and 8-nucleotide Sylamer landscape plots for all microarray experiments carried out in this study. We have highlighted the words corresponding to the miRNA seed match sites as well as the words complementary to the antimiR or LNA scramble control sequence used (see legend to Supplementary Fig. 11, exemplified by miR-21). We find that treatment with all tiny antimiRs give rise to enrichment of mRNAs containing a word complementary to the seed of the miRNA that we intended to antagonize (the miR-signature). This demonstrates functional inhibition of the targeted microRNAs. On the other hand, no effect is seen for words complementary to the antimiR or LNA scramble control oligonucleotide used. This indicates that the levels of mRNAs with binding sites for tiny antimiRs are not affected by the presence of such oligonucleotides. It should be noted that for the in vitro data on human cell lines, we observe compositional biases1 that cannot be completely corrected for by the Sylamer program.

Proteomics PC3 cells (6x106) were seeded, transfected with 25 nM antilet-7 or LNA control and harvested 48 hours post transfection. The cells were washed in ice-cold PBS and then immediately frozen at -80°C. Washed cell pellets were resuspended in 1-2 ml 8M urea, 50mM Triethylammonium bicarbonate (TEAB, pH 8.5) 0.05% w/v ProteaseMax (Promega) containing phosphatase inhibitor and protease inhibitor cocktails (P2850, P5726, P8340, Sigma) and lysed by passing three times through a 21-gauge needle and another three passes through a 25-gauge needle, on ice. The lysates were centrifuged at 14,000g, supernatants collected and protein concentrations determined by the Bicinconinic acid assay. Proteins were extracted from mouse liver samples as described2. Aliquots of 100µg total protein from each sample were reduced and alkylated by 5mM Tris(2-carboxyethyl)phosphine and 10mM Methylmethanethiosulfonate sequentially3. The reaction mixtures were then precipitated by the addition of methanol/chloroform (2:1 v/v) and the pellets reconstituted in 50µl 6M urea/50mM TEAB with sonication. An additional 50µl of 50mM TEAB containing trypsin (final ratio 1:50 w/w enzyme/protein) was added and digestion was allowed to proceed overnight at 37°C. An additional 2 hour incubation with a fresh aliquot of trypsin (1:200 w/w) was performed the next morning. The solution volumes were reduced to a final volume of ~20µl in a speed-vac, and 20µl of 1M TEAB solution added to each. iTRAQ reagents (Applied Biosystems) were added in 70µl EtOH and incubated as described3. After labeling, each solution was acidified by the addition of 3µl TFA, the separate fractions combined, and the total mixture dried in vacuo and reconstituted in 0.1% v/v TFA, 5% v/v acetonitrile for cleanup with C18 Sep-Pak cartridges (WAT036805, Waters Corp.). Bound peptides were eluted with 60% v/v acetonitrile/0.1% v/v TFA, dried, and reconstituted in OFFGEL sampling buffer in which the glycerol content was reduced to 3% v/v48. High resolution (24-fraction) OFFGEL fractionation was carried out according to the manufacturer’s protocol3. Fractionated peptides were collected (~150µl in each well) and acidified by the addition of 10µl of 5% v/v formic acid/30% v/v acetonitrile. 12µl


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of from each of the 24 fractions was injected by a Proxeon Nano LC coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo). Peptides were enriched and desalted on a homemade 5cm capillary column (250µm ID) packed with 5µm Aqua C18 material and separated on a 14cm analytical column packed with 3µm Aqua C18 material. Peptides were eluted with a gradient from 5-45% B (90% Acetonitrile, 0.1% formic acid) for 60 minutes, 45% to 80% B for 15 minutes and 80% B for 8 minutes, at a flow rate of 300nl/min. Survey full-scan spectra were acquired in an Orbitrap analyzer with resolution of 15,000 and mass range from 400-1800 m/z. Ion selection threshold was 500 counts for MS/MS. The top 4 most intense ions were selected for consecutive High-energy collisional dissociation (HCD) and Collision-induced dissociation (CID) scans. Acquisition parameters for HCD were set at a resolution of 7,500, isolation width of 2 Da, normalized collision energy at 45, activation time of 40 ms. Acquisition parameters for CID were as follows: normalized collision energy at 35, activation Q at 0.25 and an activation time of 30 ms. Dynamic exclusion settings were: repeat count of 1 in 30sec, excluded for 120sec, with exclusion list size 500. Target AGC values for the linear ion trap and orbitrap were essentially as described4.

Peaklist files were generated by Distiller (Matrix Science) where the corresponding HCD and CID scans were combined using the time-domain option. Protein identification and quantification was carried out with Mascot 2.3 (Matrix science) against the human IPI database (87,040 sequences) Methylthiolation of cysteine, N-terminal and lysine iTRAQ modifications were set as fixed modifications, methionine oxidation as a variable modification and one missed cleavage allowed. Peptide mass tolerance was set at 20 ppm, with 0.6 Da for fragment ions (decoy database false discovery rate of 1.24%). iTRAQ ratios were calculated as intensity weighted, using only peptides with expectation values < 0.05. Global ratio normalization was performed using intensity summation, with no outlier rejection.


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SUPPLEMENTARY FIGURES

Supplementary Figure 1. Inhibition of miR-21 function by tiny seed-targeting LNA. (a) Relative luciferase activity of a miR-21 luciferase reporter containing a miR-21 perfect match target site or a miR-21 mismatch target site (mismatch reporter) with two mismatches in the seed region transfected into HeLa cells. Error bars represent s.e.m. (b) Relative luciferase activity of a miR-21 luciferase reporter containing a perfect match miR-21 target site co-transfected into HepG2 cells with 1 or 5 nM tiny LNA-antimiR-21 or LNA scramble control, respectively. Error bars represent s.e.m. (c) Colony formation assay for HepG2 cells transfected with 25 nM tiny antimiR-21 or LNA scramble control. Error bars represent s.e.m.

psiCHECK2

miR-21reporter

mismatchreporter

0.0

0.5

1.0

1.5

Relative luc activity

a

b

- 1 nM 5 nM 1 nM 5 nM 0.0

0.5

1.0

1.5


antimiR-21 LNA control

psiCHECK2 miR-21 reporter

c

mock antimiR-21

LNA control

0.0

0.5

1.0

1.5

Colony formation relative to mock

Renilla luciferase ORF3´5´psiCHECK2

3´5´

3´5´

miR-21 pmt

miR-21 mm

miR-21 reporter

mismatch reporter

NNNNNN NN NNNNTCAACATCAGTCTGATAAGCTA

NNNNNN NN NNNNTCAACATCAGTCTGATCAGATA


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Supplementary Figure 2. Assessment of specificity and optimal length of LNA-antimiRs targeting the miR-21 seed region. Relative luciferase activity of a miR-21 luciferase reporter containing a miR-21 perfect match target site co-transfected into HeLa cells, with (a) 5 nM tiny seed-targeting LNA harbouring single or two adjacent mismatches at all possible nucleotide positions in the antimiR-21 sequence (highlighted in red) or (b) 1 or 5 nM of 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 12-mer LNA-antimiR. Error bars represent s.e.m.

5´- G A T A A G C T - 3´

C

C

C

C

C

C

C

G

A G

T A

A T

T T

G A

C G

T C

miR-21 reporter

psiCHECK2

seed 0.0 0.5 1.0 1.5


3´- C U A U U C G A - 5´

a

0 nM

1 nM

5 nM


6-mer 7-mer 8-mer 9-mer 10-mer 12-mer

0.0

0.5

1.0

1.5

miR-21 reporterpsiCHECK2

antimiR-21

b


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Supplementary Figure 3. Subcellular localization of antimiR-21 in HEK293 cells. A FAM-labelled antimiR-21 was delivered by unassisted uptake to HEK293 cells stably expressing a FLAG-tagged Ago2 protein. Immunofluorescence microscopy of the fixed HEK293 cells revealed a punctuated distribution for the FAM-labelled antimiR-21 (green), predominantly in the cytoplasm. The nuclei were stained with 4’,6’-diamidino-2-phenylindole (DAPI, blue) and IgG staining was used as control. Scale bar is 10µm.

Supplementary Figure 4. Specificity of tiny antimiR-122 and antimiR-155. Left panels; Relative luciferase activity of a AldoA 3’ UTR luciferase reporter co-transfected into HeLa cells with pre-miR-122, and tiny antimir-122, tiny LNA-antimiR-155, or LNA scramble control at the indicated concentrations. Right panels; Relative luciferase activity of a miR-155 luciferase reporter containing a perfect match miR-155 target site co-transfected into RAW264.7 cells with pre-miR-155, and tiny antimiR-122, tiny LNA-antimiR-155 or LNA scramble control at the indicated concentrations. Error bars represent s.e.m.

IgG DAPIantimiR-21 Merge

miR-155 reporter AldoA 3´UTR reporter


antimiR-122

antimiR-155

0.5

1.0

0.5

1.0

0 1 5

concentration (nM)

0 1 5

LNA control

0.5

1.0

- -


8

Supplementary Figure 5. Inhibition of miR-17, miR-18 and miR-19 families by 8-mer tiny LNAs. Relative luciferase activity of (a) a miR-17 luciferase reporter (b) miR-18a luciferase reporter, and (c) a miR-19a luciferase reporter, respectively, containing perfect match target sites for their cognate miRNAs, co-transfected with 1, 5 or 25 nM of (a) tiny antimiR-17 (b) antimiR-18 or (c) antimiR-19 or LNA scramble control. Error bars represent s.e.m.

0.0

0.5

1.0

1.5


0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5


nM1 5 25


-


1 5 25


nM1 5 25


- 1 5 25

psiCHECK2 miR-18a reporter

nM1 5 25


- 1 5 25

psiCHECK2 miR-19a reporter

a

b

c


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Supplementary Figure 6. Silencing of the let-7 miRNA family by seed-targeting tiny LNA in PC3 cells. (a) Relative luciferase activity of a HMGA2 3’ UTR luciferase reporter co-transfected into PC3 cells with 25 nM tiny antilet-7 or LNA scramble control (b) Western blot analysis of HMGA2 expression in PC3 cells transfected with 5 or 25 nM tiny antilet-7 or LNA scramble control. Tubulin is shown as loading control. (c) Derepression of PC3 mRNAs with canonical let-7 seed match sites after treatment with 25 nM antilet-7. The cumulative fraction plots show the distribution of log2 fold changes between the antilet-7 and LNA control treated PC3 cells for each seed match type. Kolmogorov-Smirnov tests were used to compare the three let-7 seed match types to mRNAs with no seed sites in the 3′ UTR.

Cumulative fraction

0.0

0.2

0.4

0.6

0.8

1.0

log2 fold change

6-mer

7-mer

8-mer

no site

-15

-26

a

b

c

HMGA2

Tubulin

antilet-7

mock 5 25 5 25 nM

LNA control

1

2


antilet-7

25 nM

LNA

control


10

Supplementary Figure 7. Uptake of 35S-labeled antimiR-21 in mouse tissues over time after systemic administration of a single intravenous dose of 10 mg/kg.

Time (days)

[35k

3

4

3

4

3

4

3

4

3

4

3

4

3

4

3

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4

Adrenal gl

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!!! !!

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Brain

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Heart blood

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My

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ry gland

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Thyroid gland

!!! !

!!

!!!

Uvea of the eye

!!!!

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5

Bile

!

!

!!

!!

!!

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Brown fat

! !!!

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!

!!

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Kidney ctx

!

!

!! !!!

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L

!!! !!! !!!

Ovary

!

!!

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!!

!!

Salivary gland

!!!

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Urine

!

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5

Bone marrow

!!

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Gastric m

!!

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Liver

!!! !!! !!

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L

!!!

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Pancreas

! !!!! !!!

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Sk m

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Thym

!

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!!

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Uter

!

!!

!

!

! !!

!

5Time (days) Time (days)


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Supplementary Figure 8. Targeting of miR-21 in a mouse breast tumor model by tiny antimiR-21. (a) Quantification of luciferase activity in breast tumors of antimiR-21 or LNA control injected mice 11 days after last injection. (b) Representative images of miR-21 luciferase reporter derepression in breast tumor (right side tumor) after treatment with antimiR-21 (left) or LNA control (right). Left side tumors express luciferase alone. In vivo imaging was carried out 14 days (D14) after the last dose. (c) In vivo image analysis quantification of two independent experiments. Luciferase activity was normalized to tumor size.

a

b

A S

1.50E+04

1.00E+04

5.00E+03

0.00E+00

Luciferase Activity

(counts)

0.00E+00

1.00E+05

2.00E+05

anti-miR-21

LNA control

D14

antimiR-21treated

LNA controltreated

Color BarMin = 200

Max = 8000

8000

6000

4000

2000

D14

Luciferase Activity

(counts)

c

n.s.


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Supplementary Figure 9. Assessment of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice. Mice were treated with three i.v. doses of 20 mg/kg 8-mer antimiR-122, 15-mer antimiR-122, LNA scramble control or with saline vehicle control. All values are within the normal ranges (indicated by dashed lines) for AST and ALT, respectively; n=5 per group. p<0.01 for AST levels between 8-mer antimiR-122 and LNA control treated mice, and p<0.05 for ALT levels between 15-mer antimir-122 and LNA control treated mice (one-way ANOVA).

ALT (U/L)

saline 15-mer8-mer LNA control0

25

50

75

100

antimiR-122

AST (U/L)

saline 8-mer 15-mer LNA control0

100

200

300

antimiR-122


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Supplementary Figure 10. The impact of phosphorothioate backbone modifications on in vivo silencing of miR-122 in the mouse liver by tiny seed-targeting LNA. (a) The impact of phosphorothioate backbone modifications on binding of tiny antimiR-122 to human serum albumin. The tiny antimiR-122 compounds were synthesized with a complete phosphorothioate backbone (PS) or with an unmodified phosphodiester (PO) backbone (b) Northern blot analysis of liver RNA from mice treated with three i.v. injections at 20 mg/kg of tiny antimiR-122, synthesized with a complete PS backbone or with an unmodified PO backbone or with saline. The northern blot was probed for miR-122 and U6 is shown as loading control. (c) Derepression of the direct miR-122 targets AldoA and Bckdk (same samples as in (b), normalized to GAPDH; error bars represent s.e.m. n=5), ***p<0.001 (one-way ANOVA). (d) Total plasma cholesterol levels in mice treated with three i.v. injections at 20 mg/kg of tiny antimiR-122, synthesized with a complete PS backbone or with an unmodified PO backbone or with saline (error bars represent s.e.m., n=5), ***p<0.001 (one-way ANOVA).

a

b

U6

salin

e

antimiR-122

PS PS PO

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25

50

75

100

125

Plasma cholesterol (%of saline)

saline

n.s.

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d

PS

PO

anti-miR-122

***

POsalin

e

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1

2

3

4

fold de-repression

(qRT-PCR)

BckdkAldoA

saline

PS

PO

anti-miR-122

saline

PS

PO

anti-miR-122

n.s.

***

***

n.s.

0

20

40

60

80

100

!

! !

!!

!

10 20 30 40 50 60

antimiR-122

! PO

PS


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Supplementary Figure 11. Effect of tiny LNA mediated inhibition of miR-21 and let-7 in vitro. Sylamer analyses were performed on microarray expression data from antimiR-21, antilet-7 or LNA scramble control treated cells. Shown are Sylamer enrichment landscape plots for 6-, 7-, and 8-nucleotide sites. The highlighted words in the plots correspond to canonical miR-21 and let-7 seed match sites and to perfect match binding sites for the tiny antimiR-21, antilet-7 and LNA scramble control oligonucleotides, respectively. The various types of 6-, 7- and 8-nt matches have been exemplified for miR-21 and tiny antimiR-21.


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Supplementary Figure 12. Effect of tiny LNA mediated silencing of let-7 and miR-122 on the mouse liver transcriptome. Sylamer analyses were performed on microarray expression data from antimiR-122, antilet-7 or LNA scramble control treated mouse livers. Shown are Sylamer enrichment landscape plots for 6-, 7-, and 8-nucleotide sites. The highlighted words in the plots correspond to canonical let-7 and miR-122 seed match sites and to perfect match binding sites for the tiny antilet-7, antimiR-122 and LNA scramble control, respectively.


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Supplementary Figure 13. Proteomic analysis of PC3 cells transfected with tiny antilet-7. Derepression of proteins encoded by mRNAs with canonical let-7 seed match sites after transfection with 25 nM tiny antilet-7. The cumulative fraction plots show the distribution of log2 fold changes between the antilet-7 and LNA scramble control treated cells for each seed match type. Proteins encoded by mRNAs with perfect match binding sites to antilet-7 or LNA scramble control do not show a significant shift relative to mock control.

Supplementary Figure 14. Inhibition of (a) let-7 and (b) miR-155 function by 8-mer tiny LNAs. Relative activity of the (a) HMGA2 3’ UTR and (b) miR-155 luciferase reporters, respectively, co-transfected with 5 nM tiny 8 nt LNAs tiled across the mature let-7 or miR-155 recognition sequences. Error bars represent s.e.m.

Seed sites PM binding sites

Cumulative fraction

0.0

0.2

0.4

0.6

0.8

1.0

log2 fold change0.0 0.4

6-mer7-mer8-merno site

p = 0.62p = 0.01p = 0.02

Tiny antilet-7 vs mock

2log fold change0.0 0.4

PM binding site

no sitep = 0.39

2log fold change0.0 0.4

PM binding site

no sitep = 0.61

Scrambled vs mock

a b

seed 1/2 seed center 3´ end

0.0

0.5

1.0

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0 nM

5 nM



antimiR-155

seed 1/2 seed center 3´end

0.0

0.5

1.0

1.5


HMGA2 3´ UTR reporter

0 nM

5 nM

antilet-7


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SUPPLEMENTARY TABLES

Supplementary Table 1. AntimiR oligonucleotide sequences. Oligonucleotide Sequence (5’-3’) Tm (°C)

antimiR-21 (1/2 seed) TCTGATAA 57

antimiR-21 (center) ATCAGTCT 72

antimiR-21 (3’-end) TCAACATC 49

antimiR-155 (1/2 seed) AATTAGCA 68

antimiR-155 (center) ATCACAAT 59

antimiR-155 (3’-end) CCCCTATC 64

antilet-7 (1/2 seed) ACCTACTA 70

antilet-7 (center) TACAACCT 62

antilet-7 (3’-end) AACTATAC 45

antimiR-21 (6 nt) TAAGCT 32

antimiR-21 (7 nt) ATAAGCT 45

antimiR-21 (9 nt) TGATAAGCT 68

antimiR-21 (10 nt) CTGATAAGCT 76

antimiR-21 (12 nt) GTCTGATAAGCT 87

LNA scramble (7 nt) GTAGACT <20

antimiR-21.nt1.1mm CATAAGCT 50

antimiR-21.nt2.1mm GCTAAGCT 32

antimiR-21.nt3.1mm GACAAGCT 43

antimiR-21.nt4.1mm GATCAGCT 34

antimiR-21.nt5.1mm GATACGCT 48

antimiR-21.nt6.1mm GATAACCT 32

antimiR-21.nt7.1mm GATAAGGT 50

antimiR-21.nt8.1mm GATAAGCC 64

antimiR-21.nt1.2mm AGTAAGCT 47

antimiR-21.nt2.2mm GTAAAGCT 32

antimiR-21.nt3.2mm GAATAGCT 32

antimiR-21.nt4.2mm GATTTGCT 51

antimiR-21.nt5.2mm GATAGACT 32

antimiR-21.nt6.2mm GATAACGT 33

antimiR-21.nt7.2mm GATAAGTC 51

antimiR-17 AGCACTTT 67

antimiR-18 TGCACCTT N.D.

antimiR-19 ATTTGCAC 60 LNA nucleotides are shown in uppercase. The Tm value of the 7 nt LNA scramble oligonucleotide was measured for miR-221:LNA scramble duplex. The Tm values for the antimiR-21 mismatch oligonucleotides were measured for miR-21:mismatch duplexes. N.D. = Not Determined.


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Supplementary Table 2. Repetitive human genes removed prior to Sylamer analysis. Number of

complementary sites

Number of repeats

Ensembl Gene ID Gene symbol

antimir-21

antilet-7

LNA

control

AA

GA

TGC

C

ATC

AG

GA

G

CC

TGC

TCA

TGG

AA

GA

C

TATG

CA

AC

ENSG00000124942 AHNAK 0 0 0 54 0 0 0 0 ENSG00000159692 CTBP1 0 0 0 0 41 0 0 0 ENSG00000179979 CRIPAK 0 0 0 0 0 37 0 0 ENSG00000184258 CDR1 0 0 0 0 0 0 52 0 ENSG00000185567 AHNAK2 0 0 0 43 0 1 0 0 ENSG00000205014 cDNA

FLJ50766 0 0 0 0 0 0 0 33

Supplementary Table 3. Repetitive mouse genes removed prior to Sylamer analysis. Number of sites

complementary to Number of repeats

Ensembl Gene ID Gene symbol

antimiR

-122

antilet-7

LNA

control

GC

CTG

AA

C

AA

ATA

CA

A

GG

CA

CA

TC

ENSMUSG00000006014 Prg4 1 0 0 50 0 0 ENSMUSG00000026950 Neb 0 0 0 0 25 0 ENSMUSG00000071669 Snx29 0 0 0 0 0 30


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Supplementary Table 4. Oligonucleotides used to generate miRNA luciferase reporters. Perfect match target site Strand Sequence (5´- 3´)

miR-21 Forward tcaacatcagtctgataagcta Reverse tagcttatcagactgatgttga

miR-21 Forward tcaacatcagtctgatcagata mm Reverse tatctgatcagactgatgttga

miR-155 Forward acccctatcacaattagcattaa

Reverse ttaatgctaattgtgataggggt

miR-17 Forward ctacctgcactgtaagcactttg

Reverse caaagtgcttacagtgcaggtag

miR-18a Forward ctatctgcactagatgcacctta

Reverse taaggtgcatctagtgcagatag

miR-19a Forward tcagttttgcatagatttgcaca

Reverse tgtgcaaatctatgcaaaactga

miR-221 Forward gaaacccagcagacaatgtagct

Reverse agctacattgtctgctgggtttc

miR-222 Forward gagacccagtagccagatgtagct

Reverse agctacatctggctactgggtctc

Mismatch positions are highlighted in grey

Supplementary Table 5. List of PCR primers used in this study. Target Primer Sequence (5´- 3´) AldoA 3´ UTR

Forward ccctcgagccagagctgaactaaggctgc Reverse gggcggccgcggcagtgggctggaggg

HMGA2 3´ UTR

Forward ctcgagaggtgggaggagcgaaat

Reverse gcggccgcagggttagctgcagtttgaa

Luciferase Forward cgtgtcgagatctagatccaccatggaagacgccaaaaacat

control Reverse cgtgtctcgagaattacacggcgatctttcc

miR-21 Forward cgtgtcgagatctagatccaccatggaagacgccaaaaacat

reporter Reverse cgtgtctcgagtagcttatcagactgatgttgaaattacacggcgatctttcc


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REFERENCE LIST

1. van Dongen,S., Abreu-Goodger,C., & Enright,A.J. Detecting microRNA binding and siRNA off-target effects from expression data. Nat. Methods 5, 1023-1025 (2008).

2. Worm,J. et al. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF. Nucleic Acids Res. 37, 5784-5792 (2009).

3. Ross,P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell Proteomics. 3, 1154-1169 (2004).

4. Bantscheff,M. et al. Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer. Mol. Cell Proteomics. 7, 1702-1713 (2008).


Documents

Obad et al Supp Info - Nature · PDF filecocktails (P2850, P5726, P8340, Sigma) and lysed by passing three times through a 21-gauge needle and another three passes through a 25-gauge