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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: Disney@scripps.edu

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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-

210 (200 nM). Dots represent individual miRNA gene counts (normalized intensity values).

Pearson r correlation coefficient = 0.9833.

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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:

𝐼 = 𝐼0 + 0.5∆𝜀([FL]0 − (([FL]0 + [RNA]0Kt)2 − 4[FL]0[RNA]0)

0.5) (1)

where I and I0 are the observed fluorescence intensity in the presence and absence of RNA,

respectively, is the difference between the fluorescence intensity in the absence and in the

presence of infinite RNA concentration, [FL]0 and [RNA]0 are the concentrations of compound

(Hoechst 33342 or Targapremir-210) and RNA, respectively, and Kt is the dissociation constant.

Microarray analysis of miRNA. MDA-MB-231 cells were treated with either a miRNA

antagomir against miR-210 or Targapremir-210 and placed under hypoxic conditions for 3 days.

Total RNA was isolated using the Quick-RNA MiniPrep kit (Zymo Research) and poly (A)-tailed

and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix). Biotin-labeled

RNA samples were hybridized to the GeneChip miRNA 4.0 microarray (Affymetrix). Following

hybridization, microarrays were washed and stained using the Fluidics Station 450 (Affymetrix)

and scanned using the GeneChip Scanner 3000 7G (Affymetrix). Expression analysis was

carried out using the Expression Console Software package (Affymetrix).

Tumor localization of Targapremir-210. Resected tumor samples were frozen using

O.C.T. Compound (Tissue-Tek) over dry ice. Frozen tissues were sectioned by cryostat and

transferred to microscope slides. Fluorescent microscopic analysis was carried out using a

Leica DMI3000 B upright fluorescent microscope.

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SYNTHETIC METHODS

Abbreviations: Boc, tert-butyloxycarbonyl; DCM, dichloromethane; DIC, N,N’-

Diisopropylcarbodiimide; DIEA, N,N-Diisopropylethylamine; DMF, N,N-dimethylformamide;

Fmoc, fluorenylmethoxycarbonyl chloride; HATU, 1-[Bis(dimethylamino)methylene-]1H-1,2,3-

trizaolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole;

HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser

desorption/ionization-time of flight; TFA, trifluoracetic acid

Targapremir-210

Targapremir-210 was synthesized as previously described.1

Scheme 1. Synthetic scheme for Control-CA-Biotin.

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Synthesis of Control-CA-Biotin (control Chem-CLIP probe lacking the RNA-binding

module). Rink amide resin (1 g, 0.6 mmol) was swollen in DCM for 5 min and then in DMF for

5 min. The resin was de-protected with a solution of 20% piperidine in DMF (5 mL, 2 x 20 min).

After washing with DMF (3 x 5 mL), the resin was treated with a solution of bromoacetic acid

(0.412 g, 3 mmol) and DIC (0.464 mL, 3 mmol) in DMF (5 mL) via microwave irradiation (3 x 15

s) using a 700 W microwave set to 10% power. The resin was washed with DMF (3 x 5 mL)

and then reacted with a solution of N-(4-aminoethyl)-biotin (344 mg, 1.2 mmol) and DIEA (0.3

mL, 0.6 mmol) in DMF (4 mL) via microwave irradiation (3 x 15 s) using a 700 W microwave set

to 10% power. The resin was washed with DMF (3 x 5 mL) and treated with a solution of Fmoc-

Dap(Boc)-OH (0.768 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), and DIC (0.464 mL, 3 mmol) via

microwave irradiation (3 x 15 s) using a 700 W microwave set to 10% power. The resin was

treated with a solution of 20% piperidine in DMF (4 mL), washed with DMF, and then treated

with a solution of Fmoc-β-alanine (0.56 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), DIC (0.464 mL,

3 mmol), and DIEA (0.3 mL, 1.8 mmol) in DMF (3 mL) via microwave irradiation (3 x 15 s) using

a 700 W microwave set to 10% power. The Boc protecting group was removed with a solution

of 30% TFA in DCM (4 mL) for 15 min at room temperature. The solution was concentrated in

vacuo and azeotroped with toluene three times. The resulting oil was purified by preparative

HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1% TFA over 60 min.

The purified compound (0.036 g, 0.05 mmol) was treated with a solution of acetic anhydride

(0.05 mL, 0.53 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (4 mL) via microwave irradiation (3

x 15 s) using a 700 W microwave set to 10% power. The reaction was concentrated under

vacuum and purified by preparative HPLC using a linear gradient of 20-100% CH3OH in H2O

with 0.1% TFA over 60 min. Fractions containing the product were dissolved in 20% piperidine

in DMF (2 mL) and stirred for 20 min. The reaction mixture was concentrated under vacuum

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and purified by preparative HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1%

TFA over 60 min.

The resulting purified compound was treated with a solution of chlorambucil (5 mg, 0.01 mmol),

HOAt (2.72 mg, 0.02 mmol), HATU (7.6 mg, 0.02 mmol) and DIEA (0.016 mL, 006 mmol) at

room temperature for 3 h. The mixture was then concentrated under vacuum and purified via

preparative HPLC using a linear gradient of 0-100% CH3CN in H2O with 0.1% TFA over 60 min.

Isolated 533 nmol; 0.06% yield. Retention time: 38 min; hydrolyzed product retention time: 39

min. HRMS calculated: 828.3395 (M+H), mass found: 828.4584 (M+H). Hydrolyzed product,

HRMS calculated: 792.4078 (M+H), mass found: 792.4839 (M+H). Compound can hydrolyze

during purification due to reactive alkyl halogen groups.

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Figure S8. Characterization of Control-CA-Biotin. A) Analytical HPLC trace of Control-CA-

Biotin. A linear gradient with a flow rate of 1 mL/min from 0% to 100% CH3CN + 0.1% (v/v) TFA

in water + 0.1% (v/v) TFA was used. B) HRMS MALDI-TOF of Control-CA-Biotin.

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Scheme 2. Synthetic scheme for Targapremir-210-CA-Biotin.

Synthesis of Targapremir-210-CA-Biotin (Targapremir-210 Chem-CLIP probe). Rink amide

resin (1 g, 0.6 mmol) was swollen in DCM for 5 min and then with DMF for 5 min. The resin was

de-protected with a solution of 20% piperidine in DMF (5 mL, 2 x 20 min) and then washed with

DMF (3 x 5 mL). The resin was then treated with a solution of bromoacetic acid (0.412 g, 3

mmol) and DIC (0.464 mL, 3 mmol) in DMF (5 mL) via microwave irradiation (3 x 15 s) using a

700 W microwave set to 10% power. The resin was washed with DMF (3 x 5 mL) and treated

with a solution of N-(4-aminoethyl)-biotin (344 mg, 1.2 mmol) and DIEA (0.3 mL, 0.6 mmol) in

DMF (4 mL) via microwave irradiation (3 x 15 s) using a 700 W microwave set to 10% power.

The resin was washed with DMF (3 x 5 mL) and treated with a solution of Fmoc-Dap(Boc)-OH

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(0.768 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), and DIC (0.464 mL, 3 mmol) via microwave

irradiation (3 x 15 s) using a 700 W microwave set to 10% power. The resin was treated with a

solution of 20% piperidine in DMF (4 mL), washed with DMF, and treated with a solution of

Fmoc-β-alanine (0.56 g, 1.8 mmol), HOAt (0.25 g, 1.8 mmol), DIC (0.464 mL, 3 mmol), and

DIEA (0.3 mL, 1.8 mmol) in DMF (3 mL) via microwave irradiation (3 x 15 s) using a 700 W

microwave set to 10% power. After washing with DMF, Boc was removed with a solution of

30% TFA in DCM (4 mL) for 15 min at room temperature. The solution was concentrated in

vacuo and azeotroped with toluene three times. The resulting oil was purified by preparative

HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1% TFA over 60 min.

The purified amine compound (0.036 g, 0.05 mmol) was treated with Hoechst carboxylate

(0.077 g, 0.15 mmol), HOAt (0.0204 g, 0.15 mmol), DIC (0.235 mL, 0.15 mmol) in DMF (3 mL)

in a microwave vial and reacted at 75 °C for 30 min in a Biotage Initiator. The reaction was

concentrated under vacuum and purified by preparative HPLC using a linear gradient of 20-

100% CH3OH in H2O with 0.1% TFA over 60 min.

Fractions containing the Hoechst-coupled product were re-suspended in 20% piperidine in DMF

(2 mL) and stirred for 20 min. The reaction mixture was concentrated under vacuum and

purified by preparative HPLC using a linear gradient of 20-100% CH3OH in H2O with 0.1% TFA

over 60 min.

Finally, the resulting compound was treated with a solution of chlorambucil (5 mg, 0.01 mmol),

HOAt (2.72 mg, 0.02 mmol), HATU (7.6 mg, 0.02 mmol) and DIEA (0.016 mL, 0.06 mmol) and

at room temperature for 3 h. The resulting mixture was concentrated under vacuum and

purified via preparative HPLC using a linear gradient of 0-100% CH3CN in H2O with 0.1% TFA

over 60 min. Isolated 230 nmol; 0.03% yield. Retention time: 40.5 min, HRMS calculated:

1278.5563 (M+H), mass found: 1278.7928 (M+H).

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Figure S9. Characterization of Targapremir-210-CA-Biotin. A) Analytical HPLC trace of

Targapremir-210-CA-Biotin. A linear gradient with a flow rate of 1 mL/min from 0% to 100%

CH3CN + 0.1% (v/v) TFA in water + 0.1% (v/v) TFA was used. B) HRMS MALDI-TOF of

Targapremir-210-CA-Biotin.

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REFERENCES [1] Velagapudi, S. P., Seedhouse, S. J., French, J., and Disney, M. D. J. Am. Chem. Soc.

2011; 133:10111.