Long-term, efficient inhibition of microrna function in mice using raav vectors

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1 Nature Methods Long-term, efficient inhibition of microrna function in mice using raav vectors Jun Xie, Stefan L Ameres, Randall Friedline, Jui-Hung Hung, Yu Zhang, Qing Xie, Li Zhong, Qin Su, Ran He, Mengxin Li, Huapeng Li, Xin Mu, Hongwei Zhang, Jennifer A Broderick, Jason K Kim, Zhiping Weng, Terence R Flotte, Phillip D Zamore & Guangping Gao Supplementary Figure 1 Structure of Tough Decoy (TuD) RNA. Supplementary Figure Western blot analysis of transfected HeLa cells. Supplementary Figure 3 raav genome copies per cell. Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Northern hybridization analyses. Length distribution and abundance of genome- and prefixmatching reads. Abundance of mirnas measured by high throughput sequencing. Comparison of assays to measure mirna levels. Supplementary Figure 8 Western blot analyses. Supplementary Figure 9 Body weight of the study mice. Supplementary Table 1 Oligonucleotides used in this study. Supplementary Table Sequencing Statistics: Analysis of genome-matching reads. Supplementary Table 3 Supplementary Note 1 Sequencing Statistics: Analysis of prefix-matching reads. Comparison of assays to measure mirna abundance. Nature Methods: doi:1.138/nmeth.193

2 Supplementary Figure 1 Structure of the Tough Decoy (TuD) RNA 3ʹ 5ʹ GACGGCGCUAGGAUCAUC AACACAAACACCAUUGAUCUUCACACUCCA CAA GUAUUCUG G U (U)CUGCCGCGAUCCUAGUAG -4 AAC ACCUCACACUUCUAGUUACCACAAACA CAA CAUAAGAC A C Stem 1 mirna binding site Stem TuD RNAs contain two single-stranded mirna-binding sites (here, for mir-1) flanked by double-stranded stems intended to enhance stability and promote nuclear export. Nature Methods: doi:1.138/nmeth.193

3 Supplementary Figure Western blot analysis of transfected HeLa cells Dicer Sponge Anti-let-7 TuD AntimiR-1 TuD Control GAPDH Western blot analysis of HeLa cells transfected with plasmid constructs expressing either a TuD targeting mir-1 or let-7, an antilet-7 sponge or a control. Three biological replicates are shown; Fig. c reports the quantification of these data. Nature Methods: doi:1.138/nmeth.193 3

4 Supplementary Figure 3 raav genome copies per cell AAV genome cpoies P <.1 4 weeks 5 weeks 4 weeks N. S. 5 weeks Mock AntimiR-1 TuD raav genome copies per cell in livers of C57BL/6J mice four and 5 weeks after injection with genome copies of scaav9cbgpluc (mock) or scaav9cbgpluctudmir-1 (anti-mir-1 TuD). Data are presented as mean ± standard deviation with individual values shown as filled circles. Nature Methods: doi:1.138/nmeth.193 4

5 Supplementary Figure 4 Northern hybridization analyses TuD targeting: PBS Control mir-1 let-7 Mouse: let-7 mir-1 mir-6a mir- U6 snrna Northern hybridization analyses of let-7, mir-1, mir-6a, mir- and U6 small nuclear RNA (U6 snrna) in total RNA from livers of C57BL/6J mice injected with genome copies of scaav9cbgpluc (mock), scaav9cbgpluctudmir-1 (antimir-1 TuD) or scaav9cbgpluctudlet-7 (anti-let-7 TuD). The animals were sacrificed four weeks after injection and total liver RNA from three biological replicates measured to obtain the correction factor used in high throughput sequencing analyses. Nature Methods: doi:1.138/nmeth.193 5

6 Supplementary Figure 5 Length distribution and abundance of genome- or prefix-matching reads All let-7 isoforms let-7e 5 -UGAGGUAGGA GGUUGUAUAGUU-3 Normalized genomematching reads (x 1 3 ) Normalized genomematching reads (x 1 3 ) Normalized genomematching reads (x 1 3 ) reads (x 1 3 ) let-7a 5 -UGAGGUAGUA GGUUGUAUAGUU-3 let-7f 5 -UGAGGUAGUA GAUUGUAUAGUU-3 reads (x 1 3 ) reads (x 1 3 ) A (5%) AA (8%) AAA(1%) A (73%) C (13%) AA (5%) A (94%) G (3%) C (%) Normalized genomematching reads (x 1 3 ) reads (x 1 3 ) 3 1 AA (53%) A (3%) C (7%) A (71%) C (11%) AA (6%) A (9%) AA (6%) C (1%) let-7b 5 -UGAGGUAGUA GGUUGUGUGGUU-3 : : let-7g 5 -UGAGGUAGUA GUUUGUACAGUU-3 reads (x 1 3 ) reads (x 1 3 ) Normalized genomematching reads (x 1 3 ) reads (x 1 3 ) let-7c 5 -UGAGGUAGUA GGUUGUAUGGUU-3 : let-7i 5 -UGAGGUAGUA GUUUGUGCUGUU-3 : reads (x 1 3 ) reads (x 1 3 ) A (57%) AA (33%) AG (%) A (6%) AA (18%) C (7%) A (83%) G (4%) AAA (3%) Normalized genomematching reads (x 1 3 ) reads (x 1 3 ) let-7d 5 -UGAGGUAGUA GGUUGCAUAGUU-3 mir-98 TuD 5 -UGAGGUAGUAA GUUGUAUUGUU-3 3 -ACUCCAUCAUU CUA C CAACAUAUCAA-5 reads (x 1 3 ) reads (x 1 3 ).6.4. A (57%) AA (9%) C (3%) A (65%) C (14%) AA (7%) A (79%) AA (1%) C (%) Normalized genomematching reads (x 1 3 ) reads (x 1 3 ) A (67%) C (17%) AU (16%) A (83%) C (17%) U (63%) A (31%) AA (3%) U (51%) A (36%) AAU (6%) Length distribution and abundance of genome-matching or prefix-matching let-7 paralog sequence reads in livers of mice four weeks after injection of scaav9cbgpluc (mock), scaav9cbgpluctud let-7 (anti-let-7 TuD). The most abundant non-templated nucleotides added to the 3 end of the let-7 isoforms are indicated in the grey boxes. Nature Methods: doi:1.138/nmeth.193 6

7 Supplementary Figure 6 Abundance of mirnas measured by high throughput sequencing Anti-miR-1 TuD (ppm) 1,48,576 3,768 1,4 r =.94 P <.1 mir-1 3 Anti-let-7 TuD (corr. ppm) 1,48,576 3,768 1,4 let-7e let-7b let-7i mir-98 let-7d let-7g let-7f let-7a let-7c 3 3 1,4 3,768 1,48,576 Control (ppm) Abundance of mirnas in liver of mice 4 weeks after injection of scaav9cbgpluc (mock), scaav9cbgpluctud let-7 (anti-let-7 TuD) or scaav9cbgpluctudmir-1 (anti-mir-1 TuD). Pearson correlation analysis was performed using GraphPad Prism V5.b (GraphPad Software, Inc.). The correlation coefficient (r) and P-value are indicated. mirnas targeted by TuDs are red. Nature Methods: doi:1.138/nmeth.193 7

8 Supplementary Figure 7 Comparison of assays used to measure mirna levels qrt-pcr Northern hybridization High throughput sequencing mir-1 abundance Control Anti-miR-1 TuD Prefixmatching Genomematching Apparent reduction: 83% 8% 77% 19% Full length mir-1 All mir-1 isoforms Comparison of assays used to measure mir-1 in Fig. 3. Nature Methods: doi:1.138/nmeth.193 8

9 Supplementary Figure 8 Western blot analyses TuD targeting: CyclinG1 Control mir-1 let Dicer GAPDH HFE TMED3 GAPDH Ras GAPDH Western blot analysis of mouse livers samples four weeks after injection of scaav9cbgpluc (Control), scaav9cbgpluctudmir-1 (antimir-1 TuD) or scaav9cbgpluctudlet-7 (anti-let-7 TuD). Nature Methods: doi:1.138/nmeth.193 9

10 Supplementary Figure 9 Body weight of the study mice. Weight (g) Anti-miR-1 TuD (n = 4) Anti-let-7 TuD (n = 3) Control (n = 4) 1 15 Weeks after injection 5 The C57BL/6J mice treated with genome copies of scaav9cbgpluc (control), scaav9cbgpluctudmir-1 (anti-mir-1 TuD) or scaav9cbgpluctudlet-7 (anti-let-7 TuD) were weighed 1, 1, 14, 16, 18, and 5 weeks after via tail vein injection. Mean ± standard deviation is shown. Nature Methods: doi:1.138/nmeth.193 1

11 Supplementary Table 1 Oligonucleotides used in this study. Oligonucleotide Sequence (5! to 3!) anti-mir-1 TuD anti-let-7 TuD mir-1 mirzip anti-let-7 mirzip anti-mir-1 sponge anti-let-7 sponge Mutant anti-mir-1 sponge Mutant anti-let-7 sponge (mir-1) 1 sense (mir-1) 1 anti-sense (mir-1) 3 sense (mir-1) 3 anti-sense XbaI ApaI linker F XbaI ApaI linker R pri-mir-1 F pri-mir-1 R Nras F Nras R Kras F Kras R GGATCCGACGGCGCTAGGATCATCAACCAAACACCATTGATCTTCACACTC CACAAGTATTCTGGTCACAGAATACAACCAAACACCATTGATCTTCACACT CCACAAGATGATCCTAGCGCCGTCTTTTTTGAATTC GGATCCGACGGCGCTAGGATCATCAACAACTATACAACCATCTTACTACCT CACAAGTATTCTGGTCACAGAATACAACAACTATACAACCATCTTACTACC TCACAAGATGATCCTAGCGCCGTCTTTTTTGAATTC GGATCCTGGTCAGTGACAATGTTTGCTTCCTGTCAGACAAACACCATTGTC ACACTCCATTTTTAAGCTTGAAGACAATAGC GGATCCTCTCGTAGTAGGTTGTATAGTTCTTCCTGTCAGAAACTATACAAC CTACTACCTCATTTTTAAGCTTGAAGACAATAGC TCTAGACAAACACCATACAACACTCCACAAACACCATACAACACTCCACAA ACACCATACAACACTCCACAAACACCATACAACACTCCACAAACACCATAC AACACTCCACAAACACCATACAACACTCCACAAACACCATACAACACTCCA GGGCCC TCTAGAAACTATACAAAACCTACCTCAAACCACACAAAACCTACCTCAAAC CATACAAAACCTACCTCAAACTATGCAAAACCTACCTCTAACTATACAAAA CCTACCTCAAACTGTACAAAACCTACCTCAAACCATACAAAACCTACCTCA GGGCCC TCTAGACAAACACCATACAACAAGAAACAAACACCATACAACAAGAAACAA ACACCATACAACAAGAAACAAACACCATACAACAAGAAACAAACACCATAC AACAAGAAACAAACACCATACAACAAGAAACAAACACCATACAACAAGAAA GGGCCC TCTAGAAACTATACAAAACCTAAAGAAAACCACACAAAACCTAAAGAAAAC CATACAAAACCTAAAGAAAACTATGCAAAACCTAAAGATAACTATACAAAA CCTAAAGAAAACTGTACAAAACCTAAAGAAAACCATACAAAACCTAAAGAA GGGCCC pcgaaacaaacaccattgtcacactccatt pcgaatggagtgtgacaatggtgtttgttt pcgaaacaaacaccattgtcacactccaacaaacaccattgtcacactcca ACAAACACCATTGTCACACTCCATT pcgaatggagtgtgacaatggtgtttgttggagtgtgacaatggtgtttgt TGGAGTGTGACAATGGTGTTTGTTT CTAGATTCCGAGATATCGGTAATGGGCC GGCCCATTACCGATATCTCGGAATCTAG ATCGGGCCCGACTGCAGTTTCAGCGTTTG CGCGGGCCCGACTTTACATTACACACAAT TGGACACAGCTGGACAAGAG CTGTCCTTGTTGGCAAGTCA CAAGAGCGCCTTGACGATACA CCAAGAGACAGGTTTCTCCATC! Nature Methods: doi:1.138/nmeth

12 Hras1 F Hras1 R Mm-Dicer F Mm-Dicer R c-myc F c-myc R Hfe F Hfe R Tmed3 F Tmed3 R Aldolase A F Aldolase A R CAT-1 F CAT-1 R Cyclin G1 F Cyclin G1 R Mm-Actin F Mm-Actin R mir-1 probe Let-7 probe mir-6a probe mir- Probe U6 probe IDT mirna cloning linker-1 CGTGAGATTCGGCAGCATAAA GACAGCACACATTTGCAGCTC GCAGGCTTTTTACACACGCCT GGGTCTTCATAAAGGTGCTT CAACGTCTTGGAACGTCAGA TCGTCTGCTTGAATGGACAG GGGGACCTTGCTTTCCACTC GCCTCATAGTCACAGGGATCT AGCAGGGCGTGAAGTTCTC TTGTACGTGAAGCTGTCATACTG TGGGAAGAAGGAGAACCTGA AGTGTTGATGGAGCAGCCTT TACCAGTGGCCGTGTTTGTA GCTGTTGCCAAGCTTCTACC AATGGCCTCAGAATGACTGC AGTCGCTTTCACAGCCAAAT ATGCCAACACAGTGCTGTCTGG TGCTTGCTGATCCACATCTGCT TGGAGTGTGACAATGGTGTTTG AACTATACAACCTACTACCTCA AGCCTATCCTGGATTACTTGAA ACAGTTCTTCAACTGGCAGCTT CTCTGTATCGTTCCAATTTTAGTATA AppCTGTAGGCACCATCAAT/ddC/ 5! Illumina RNA Adapter GUUCAGAGUUCUACAGUCCGACGAUC Small RNA RT primer Small RNA PCR Primer 1 Small RNA PCR Primer ATTGATGGTGCCTACAG CAAGCAGAAGACGGCATACGAATTGATGGTGCCTACAG AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA! Nature Methods: doi:1.138/nmeth.193 1

13 Supplementary Table Sequencing statistics: Analysis of genome-matching reads. Sample Total reads Reads perfectly matching genome Reads matching annotated ncrnas Small RNA reads excluding ncrnas Pre-miRNAmatching reads Control 3,39,64,335,379 8,894,36,485,174,544 anti-mir-1 TuD 3,386,944,189,155 16,366,17,789 1,917,478 anti-let-7 TuD 1,893,1 1,3,744 4,1 1,8,73 1,163,466 Supplementary Table 3 Sequencing statistics: Analysis of 5! prefix-matching reads. To detect small RNAs bearing 3! terminal, non-templated nucleotides, reads matching the reference genome for only part of their entire length were identified. Sample Total reads Prefixes matching genome Prefixes excluding internal mismatch Prefixes matching annotated ncrnas Prefixes excluding ncrnas PremiRNAmatching reads Control 3,39,64 93,885 8,519 1,9 798, ,87 anti-mir-1 TuD 3,386,944 1,197,478 1,86,49,855 1,83, ,181 anti-let-7 TuD 1,893,1 66,68 574,454 1,8 573, ,9!!!! Nature Methods: doi:1.138/nmeth

14 Supplementary Note 1 Comparison of assays to measure mirna abundance Quantitative reverse transcription and polymerase chain reaction (qrt-pcr), Northern hybridization and high throughput sequencing of small RNA libraries are currently the most widely used techniques to quantify changes in small RNA expression. We compared these three techniques to assess their performance in detecting changes in mirna expression directed by a TuD mirna inhibitor (Supplementary Fig. 7). qrt-pcr detected an 83% reduction in mir-1 levels in total RNA from mouse liver 4 weeks after infection with raav-tud-mir-1, compared with control mice receiving raav that did not express a TuD. High throughput sequencing of small RNA libraries generated from the same samples detected a 77% reduction in the full-length, 3 nt mir-1. We conclude that qrt-pcr detects mainly the single isoform against which the primers have been designed. It therefore provides a method to detect a specific mirna sequence in the presence of tailed and trimmed isoforms. In contrast, Northern hybridization detected only a 8% decrease in mir- 1 because multiple mir-1 isoforms were detected (Fig. 4). High throughput sequencing detected a 19% decrease in all mir-1 isoforms, including genomematching and prefix-matching, tailed reads. We conclude that Northern hybridization provides a method to assess changes in the abundance of all isoforms, including those modified by tailing or trimming. We note that Northern hybridization may detect shorter mir-1 isoforms, which increase after TuD RNA expression (Fig. 3c,d) less well than full-length mir-1, because the radiolabeled probe will make more base pairs with the longer isoform. This may explain why our Northern hybridization measurements suggest a greater decrease for mir-1 overall than is reported by our high throughput sequencing data. Finally, we note that although high throughput sequencing currently provides the most complete representation of changes in genome- and prefixmatching mirna isoforms, normalization is required to compare the change in mirna abundance between different libraries. Therefore, any change in mirna abundance between different libraries should be supported by other techniques.! Nature Methods: doi:1.138/nmeth

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