S-1 Supporting Information for: Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit Matthew G. Costales, 1,4 Christopher L. Haga, 2,4 Sai Pradeep Velagapudi, 1,4 Jessica L. Childs- Disney, 1 Donald G. Phinney, 2 and Matthew D. Disney 1,3,* 1 Department of Chemistry, 2 Department of Molecular Therapeutics, and 3 Department of Neuroscience, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458 4 These authors contributed equally. *Author to whom correspondence should be addressed: [email protected]
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Supporting Information for: Small Molecule Inhibition of ...€¦ · S-1 Supporting Information for: Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit
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Supporting Information for:
Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit
Matthew G. Costales,1,4 Christopher L. Haga,2,4 Sai Pradeep Velagapudi,1,4 Jessica L. Childs-Disney,1 Donald G. Phinney,2 and Matthew D. Disney1,3,*
1Department of Chemistry, 2Department of Molecular Therapeutics, and 3Department of
Neuroscience, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458
4These authors contributed equally.
*Author to whom correspondence should be addressed: [email protected]
SUPPLEMENTARY FIGURES & TABLE Figure S1. Binding assays of Targapremir-210 and Hoechst 33342 to pre-miR-210. A) Representative binding curve of Targapremir-210 with pre-miR-210. B) Representative binding curve of Hoechst 33342 with pre-miR-210. C) Secondary structure of pre-miR-210.
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Figure S2. Specificity of Targapremir-210 and miR-210 antagomir. MiRNA microarray analysis
of hypoxic MDA-MB-231 cells treated with either a miR-210 antagomir (50 nM) or Targapremir-
Figure S3. Studying the non-selective binding of Targapremir-210-CA-Biotin Chem-CLIP probe. A) Using a control Chem-CLIP probe that lacks the RNA-binding module, Control-CA-Biotin, we studied if highly abundant transcripts that were pulled down by Targapremir-210-CA-Biotin were due to non-specific effects from the chlorambucil (CA) and biotin modules. For three of the RNAs, there is no difference between enrichment from Targapremir-210-CA-Biotin and Control-CA-Biotin, suggesting that pull-down is due to non-selective reaction with CA. B) A C-Chem-CLIP experiment was completed to assess non-selective binding caused by addition of CA and biotin modules for hypoxia-associated miRNAs pulled down by Targapremir-210-CA-Biotin. Compared to the C-Chem-CLIP results with miR-210, the abundance of other miRNAs does not decrease as dramatically, indicating that some of the pull-down may be due to non-selective effects of the CA or biotin moieties. *, p<0.05, as compared to before pull-down fraction, as determined by a two-tailed Student t-test.
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Figure S4. Targapremir-210 (200 nM) selectively inhibits biogenesis of miR-210 in MDA-MB-231 cells cultured under hypoxic conditions while Targaprimir-96 (200 nM) selectively inhibits biogenesis of miR-96. * indicates p<0.05, as compared to the untreated sample, as determined by a two-tailed Student t-test.
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Figure S5. Studying Targapremir-210’s (200 nM) effect on in vitro topoisomerase activity. Targapremir-210 did not inhibit the formation of topoisomers (+Topo II) nor does it result in the formation of a linear product, unlike positive control VP16 (red box).
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Figure S6. Biological effect of Hoechst 33342. qPCR analysis of hypoxic MDA-MB-231 cells treated with 200 nM of either Hoechst 33342 or Targapremir-210. Targapremir-210 significantly affects biogenesis of miR-210 compared to the untreated sample. * indicates p<0.05, as determined by a two-tailed Student t-test.
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Figure S7. Tumor localization of Targapremir-210. Frozen resected tumor samples from either control, antagomir-treated, or Targapremir-210-treated mice showing localization of Targapremir-210 within the tumor mass as determined by fluorescent microscopy.
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Table S1. Sequences of qRT-PCR primers hsa-miR-210 FWD CTGTGCGTGTGACAGCGGCTGA
pre-miR-210 FWD GCAGCCCCTGCCCACCGCACACT
pre-miR-210 REV CCGCTGTCACACGCACAG
pri-miR-210 FWD GACTGGCCTTTGGAAGCTCC
pri-miR-210 REV ACAGCCTTTCTCAGGTGCAG
HIF1α FWD CGCGAACGACAAGAAAAAG
HIF1α REV AAGTGGCAACTGATGAGCAA
GPD1L FWD CCCCTGAAAGTGTGCATCGT
GPD1L REV GGCAGCTTGTGTCCAGGAA
hsa-miR-10b FWD TACCCTGTAGAACCGAATTTGTG
hsa-miR-21 FWD TAGCTTATCAGACTGATGTTGA
hsa-miR-23a FWD ATCACATTGCCAGGGATTTCC
hsa-miR-23b FWD ATCACATTGCCAGGGATTACC
hsa-miR-24 FWD TGGCTCAGTTCAGCAGGAACAG
hsa-miR-26a FWD TTCAAGTAATCCAGGATAGGCT
hsa-miR-26b FWD TTCAAGTAATTCAGGATAGGT
hsa-miR-27a FWD TTCACAGTGGCTAAGTTCCGC
hsa-miR-30a FWD TGTAAACATCCTCGACTGGAAG
hsa-miR-30b FWD TGTAAACATCCTACACTCAGCT
hsa-miR-30c FWD TGTAAACATCCTACACTCTCAGC
hsa-miR-30d FWD TGTAAACATCCCCGACTGGAAG
hsa-miR-93 FWD CAAAGTGCTGTTCGTGCAGGTAG
hsa-miR-103 FWD AGCAGCATTGTACAGGGCTATGA
hsa-miR-106a-5p FWD AAAAGTGCTTACAGTGCAGGTAG
hsa-miR-125a-5p FWD TCCCTGAGACCCTTTAACCTGTGA
hsa-miR-125b FWD TCCCTGAGACCCTAACTTGTGA
hsa-miR-152 FWD TCAGTGCATGACAGAACTTGG
hsa-miR-181a FWD AACATTCAACGCTGTCGGTGAGT
hsa-miR-192 FWD CTGACCTATGAATTGACAGCC
hsa-miR-193b FWD AACTGGCCCTCAAAGTCCCGCT
hsa-miR-195 FWD TAGCAGCACAGAAATATTGGC
hsa-miR-205 FWD TCCTTCATTCCACCGGAGTCTG
hsa-miR-206 FWD TGGAATGTAAGGAAGTGTGTGG
hsa-miR-224 FWD CAAGTCACTAGTGGTTCCGTT
hsa-miR-335 FWD TCAAGAGCAATAACGAAAAATGT
hsa-miR-339-5p FWD TCCCTGTCCTCCAGGAGCTCACG
hsa-miR-491-3p FWD CTTATGCAAGATTCCCTTCTAC
hsa-miR-497 FWD CAGCAGCACACTGTGGTTTGT
hsa-miR-466 FWD ATACACATACACGCAACACACAT
hsa-miR-505 FWD CGTCAACACTTGCTGGTTTCCT
hsa-miR-140 FWD TACCACAGGGTAGAACCACGG
hsa-miR-181a-2 FWD AACATTCAACGCTGTCGGTGAGT
hsa-miR-324 FWD ACTGCCCCAGGTGCTGCTGG
hsa-miR-648 FWD AAGTGTGCAGGGCACTGGT
hsa-miR-103a-1 FWD AGCAGCATTGTACAGGGCTATGA
hsa-miR-1273c FWD GGCGACAAAACGAGACCCTGTC
hsa-miR-4682 FWD TCTGAGTTCCTGGAGCCTGGTCT
hsa-miR-3120 FWD CACAGCAAGTGTAGACAGGCA
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hsa-miR-3174 FWD TAGTGAGTTAGAGATGCAGAGCC
hsa-miR-4446 FWD CAGGGCTGGCAGTGACATGGGT
hsa-miR-107 FWD AGCAGCATTGTACAGGGCTATCA
hsa-miR-3613 FWD ACAAAAAAAAAAGCCCAACCCTTC
RNU6 FWD ACACGCAAATTCGTGAAGCGTTC
Universal REV GAATCGAGCACCAGTTAC
18S Rrna-FWD GTAACCCGTTGAACCCCATT
18S Rrna-REV CCATCCAATCGGTAGTAGCG
28S rRNA-FWD AGAGGTAAACGGGTGGGGTC
28S rRNA-REV GGGGTCGGGAGGAACGG
5.8S rRNA-FWD ACTCGGCTCGTGCGTC
5.8S rRNA-REV GCGACGCTCAGACAGG
45S rRNA-FWD GAACGGTGGTGTGTCGTT
45S rRNA-REV GCGTCTCGTCTCGTCTCACT
5S rRNA-FWD GGCCATACCACCCTGAACGC
5S rRNA-REV CAGCACCCGGTATTCCCAGG
7SK-FWD CCCCTGCTAGAACCTCCAAAC
7SK-REV CACATGCAGCGCCTCATTT
7SL-FWD ATCGGGTGTCCGCACTAAGTT
7SL-REV CAGCACGGGAGTTTTGACCT
ACA16-FWD GGCCCTTATCGAAGCTGCA
ACA16-REV CGGCGACCGTCAAGGA
ACA44-FWD GTTTCCAAGGGCTGTGGCT
ACA44-REV TGTACTGACCTGCGCTGTCAA
ACA61-FWD CCTTTCCCATCGGATCTGAA
ACA61-REV CCACATGCCATATACCAGATTACAAC
BC200-FWD TGGCTCACGCCTGTAATCC
BC200-REV CCCAGGCAGGTCTCGAACT
HBI-36-FWD CAGCACTGCCAAGTGACCC
HBI-36-REV ATATGTACCCAGCTGCATGCAG
HBII-85-FWD TGGATCGATGATGAGTCC
HBII-85-REV TGGACCTCAGTTCCGATGAGA
HBII-420-FWD ACTGGTCCAGGATGAAACCTAATT
HBII-420-REV CCTAGGAGCTGGTCTCAGTCCC
U1-FWD CCATGATCACGAAGGTGGTTT
U1-REV ATGCAGTCGAGTTTCCCACAT
U2-FWD TTCTCGGCCTTTTGGCTAAG
U2-REV CTCCCTGCTCCAAAAATCCA
U4-FWD GCCAATGAGGTTTATCCGAGG
U4-REV TCAAAAATTGCCAATGCCG
U5-FWD TGGTTTCTCTTCAGATCGCATAAA
U5-REV CCAAGGCAAGGCTCAAAAAAT
U6-FWD CTCGCTTCGGCAGCACA
U6-REV AACGCTTCACGAATTTGCGT
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U12-FWD GCCCGAATCCTCACTGCTAA
U12-REV TCGCAACTCCCAGGCATC
U87-FWD ATGGGATCATGGAGCAGCTG
U87-REV TCACACCCATGACTGCCACT
U105-FWD CCCCTATCTCTCATGATGAACACATAT
U105-REV CCCCATCTCTTCTTCAGAGCG
tRNA(Lys)-FWD CGGCTAGCTCAGTCGGTAGA
tRNA(Lys)-REV CCCACGACCCTGAGATTAAG
tRNA(Ala)-FWD GGGGGTGTAGCTCAGTGGTA
tRNA(Ala)-REV AGGCCTCATACATGCAAAGC
tRNA(Cys)-FWD GCTCAGGGGTAGAGCATTTG
tRNA(Cys)-REV ACCGGGGACTTCTGGATCT
tRNA(His)-FWD GCAGTGACTGTATAGTGGTTAGCA
tRNA(His)-REV GTGGCCACAACACAGAGTG
tRNA(Ser)-FWD TAGTCGTGGCCGAGTGGTTA
tRNA(Ser)-REV GGAAACCCCAATGGATTTCTA
tRNA(Val)-FWD TGGTTATCACGTTCGCCTAA
tRNA(Val)-REV GTTTCGAACCGGGGACCT
tRNA(Arg)-FWD CAGTGGCGCAATGGATAAC
tRNA(Arg)-REV CAGGAGTCGAACCTGGAATC
tRNA(Gln)-FWD TGGTTAGCACTCTGGACTCTGA
tRNA(Gln)-REV AGGTTCCACCGAGATTTGAA
tRNA(Ile)-FWD CAGTTGGTTAGAGCGTGGTG
tRNA(Ile)-REV CCACGACCTTGGCGTTATTA
tRNA(Thr)-FWD TAGCTGGTTAAAGCGCCTGT
tRNA(Thr)-REV GAACCCAGGATCTCCTGTTTACT
tRNA(Asn)-FWD CAATGGGTTAGCGCGTTC
tRNA(Asn)-REV AACCACCAACCTTTCGGTTA
tRNA(Glu)-FWD CTGGTGGTCTAGTGGCTAGGA
tRNA(Glu)-REV CTGGCCGGGAATCGAAC
tRNA(Ini)-FWD CATAACCCAGAGGTCGATGG
tRNA(Ini)-REV TAGCAGAGGATGGTTTCGAT
tRNA(Phe)-FWD TCAGTTGGGAGAGCGTTACA
tRNA(Phe)-REV AGGGTTGAACCAGGGAACTT
tRNA(Trp)-FWD GCGCGTCTGACTCCAGAT
tRNA(Trp)-REV ACGTGATTTGAACACGCAAC
tRNA(Asp)-FWD TCTGCCTGTCATGTGGAGAC
tRNA(Asp)-REV CCTGTTGGGGACTCAAACTC
tRNA(Gly)-FWD GCATTGGTGGTTCAGTGGTA
tRNA(Gly)-REV ATTGGCCGGGAATTGAAC
tRNA(Leu)-FWD GGTCTAAGGCGCTGGATTAAG
tRNA(Leu)-REV CCCCCGAAGAGACTGGAG
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tRNA(Pro)-FWD GGGGTATGATTCTCGCTTAGG
tRNA(Pro)-REV ATTTGAACCCGGGACCTCT
tRNA(Tyr)-FWD CTGGTAGAGCGGAGGACTGT
tRNA(Tyr)-REV GGAATTGAACCAGCGACCTA
tRNA(Sec)-FWD GGCTTCAAACCTGTAGCTGTC
tRNA(Sec)-REV CCGAAATGGAATTGAACCAC
FTH1-FWD GGCAAAGTTCTTCAAAGCCA
FTH1-REV CATCAACCGCCAGATCAAC
GAPDH-FWD TGCACCACCAACTGCTTAGC
GAPDH-REV GGCATGGACTGTGGTCATGAG
ATP6-FWD TTCTGGAATGACTCCTTTGC
ATP6-REV TTGGCCAGAATGAACTTGAA
RPL8-FWD AGATGGGTTTGTCAATTCGG
RPL8-REV CAAGAAGACCCGTGTGAAGC
FLNA-FWD AGGGGACGGCCCTTTAAT
FLNA-REV GTCGCTCTCAGGAACAGCAG
RPS6-FWD CAAGAGAATGAAGGAGGCTAAGG
RPS6-REV AAGCTCGCAGAGAGGAAAGTCT
Rpl11-FWD ACTTCGCATCCGCAAACTCT
Rpl11-REV TGTGAGCTGCTCCAACACCTT
ENO1-FWD GCCTCCTGCTCAAAGTCAAC
ENO1-REV AACGATGAGACACCATGACG
COX2-FWD GCAGGGTTGCTGGTGGTAG
COX2-REV ATTTCATCTGCCTGCTCTGG
GNB2L1-FWD TGGCTAACTGCAAGCTGAAGAC
GNB2L1-REV ATCTGGAGAGACAGTCACCGTG
PKM2-FWD TACCATGCGGAGACCATCAA
PKM2-REV AGCAACGGGCCGGTAGAG
CLU-FWD CCAGTGGAAGATGCTCAAC
CLU-REV CGAGTCAGAAGTGTGGGAAGC
CANX-FWD GCAACCACTTCCCTTCCAT
CANX-REV TCCGCCTCTCTCTTTACTGC
RPS9-FWD AGACCCAGGTCTTCAAGCTG
RPS9-REV ATGAAGGACGGGATGTTCAC
GNAS-FWD CAGTGGAGATGGGCGTCACTA
GNAS-REV CGGCGGATGTTCTCAGTGT
COX1-FWD CGATGCATACACCACATGAA
COX1-REV AGCGAAGGCTTCTCAAATCA
B2M-FWD TGCTGTCTCCATGTTTGATGTATCT
B2M-REV TCTCTGCTCCCCACCTCTAAGT
RPS3-FWD TCCTCGGAGTTTCCCAGAC
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RPS3-REV TCCTAGGAGGGCTTGCTGT
EEF1A1-FWD TGCGGTTTTTGTCATCAAA
EEF1A1-REV AAGAGTGGGGTGGCAGGTATTAG
Rpl27-FWD TCCAAGGGGATATCCACAGA
Rpl27-REV CATGGGCAAGAAGAAGATCG
RPL37-FWD AAAACCAGAACATTTATTGCATGA
RPL37-REV TCCGTGAAGGAACAACACCT
RPL3-FWD CCTCCGTTCACCTGGATCT
RPL3-REV CCAAGTCATCCGTGTCATTG
EEF2-FWD GCACGTTCGACTCTTCACTG
EEF2-REV CTGGAGATCTGCCTGAAGGA
ND2-FWD GCCCTAGAAATAAACATGCTA
ND2-REV GGGCTATTCCTAGTTTTATT
TPT1-FWD CATGATTATCTACCGGGACCTCAT
TPT1-REV AACCCGTCCGCGATCTC
TMSB4X-FWD AGACCAGACTTCGCTCGTA
TMSB4X-REV CTGCTTCGTTCTCCTGTT
RPL4-FWD GCTCTGGCCAGGGTGCTTTTG
RPL4-REV ATGGCGTATCGTTTTTGGGTTGT
CCT5-FWD ACAGCCTTTGCTGCCATTAT
CCT5-REV GCCCTTTCCTCATCATCAAG
PRDX1-FWD GGGCACACAAAGGTGAAGTC
PRDX1-REV GCTGTTATGCCAGATGGTCAG
PFN1-FWD GATCACCGAACATTTCTGGC
PFN1-REV AAACGTTCGTCAACATCACG
Rps24-FWD AAGACCACACCGGATGTCATC
Rps24-REV TGCCAAAGCCAGTTGTCTTG
PPIA-FWD TTATTTGGGTTGCTCCCTTC
PPIA-REV AAGTGTGCCAAATCTGCAAG
LDHB-FWD TGGCGTGTGCTATCAGCATT
LDHB-REV GCTTATCTTCCAAAACATCCACAAG
LGALS1-FWD AAGCTGCCAGATGGATACGAA
LGALS1-REV CGTCAGCTGCCATGTAGTTGA
PGK1-FWD ATGGATGAGGTGGTGAAAGC
PGK1-REV CAGTGCTCACATGGCTGACT
BASP1-FWD CAATGCCAATCCTCCATTCT
BASP1-REV AACTACAGGTGCACCCAACC
YBX1-FWD TGATGGAGGGTGCTGACAAC
YBX1-REV CCTGCGGAATCGTGGTCTAT
KRT7-FWD TGGAGGACTTCAAGAATAAGTACGAA
KRT7-REV TCATGTAGGCAGCATCCACATC
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CYTB-FWD AATTCTCCGATCCGTCCCTA
CYTB-REV GGAGGATGGGGATTATTGCT
NONO-FWD CCCTTACAGTTCGAAACCTT
NONO-REV ATGACTACAGCCCTCTCTAC
ND1-FWD ATACCCCCCATTCCGCTACGAC
ND1-REV GTTTGAGGGGGAATGCTGGAGA
ALDOA-FWD CGGGAAGAAGGAGAACCTG
ALDOA-REV GACCGCTCGGAGTGTACTTT
P4HB-FWD GCAAACTGAGCAACTTCAAA
P4HB-REV TTCTTCAGGCCAAAGAACTC
LDHA-FWD ACCCAGTTTCCACCATGATT
LDHA-REV CCCAAAATGCAAGGAACACT
APP-FWD GCTGGCTGAACCCCAGATT
APP-REV CCCACTTCCCATTCTGGACAT
FOLR1-FWD GCACCACAAGGAAAAGCCAG
FOLR1-REV CATTCTTCCTCCAGGGTCGAC
ACTB-FWD CTGGAACGGTGAAGGTGACA
ACTB-REV AAGGGACTTCCTGTAACAATGCA
RPL13-FWD CCCGTCCGGAACGTCTATAA
RPL13-REV CTAGCGAAGGCTTTGAAATTCTTC
RPS21-FWD GGCGAGTTCGTGGACCTGTA
RPS21-REV GGATGGATGCGTGGTCCTT
S100A6-FWD CTGCAGGATGCTGAAATTGC
S100A6-REV GGAAGTTCACCTCCTGGTCCTT
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SUPPLEMENTARY METHODS
Determination of Binding Affinities of Hoechst 33342 and Targapremir-210.
Dissociation constants for the binding of pre-miR-210 RNA to Hoechst 33342 and Targapremir-
210 were determined using an in solution, fluorescent binding assay. RNA was folded in 1×
Binding Buffer (8 mM Na2HPO4, 190 mM NaCl, 1 mM EDTA, and 40 µg/mL BSA) at 60 °C for 5
min and then allowed to cool to room temperature for 10 min. Hoechst 33342 or Targapremir-
210 was added to a final concentration of 500 nM. Next, 1:2 serial dilutions were performed in
1× Binding Buffer supplemented with 500 nM of Hoechst 33342 or Targapremir-210. Solutions
were incubated for 30 min and then transferred to Corning non-binding surface 96-well black
plates. Fluorescence intensity was then measured on a Bio-Tek FLX-800 plate reader. Change
in fluorescence intensity was fit as a function of RNA concentration with equation 1: