Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe

Transcription

1 Supporting Information Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe Shengnan He, Long Qu, Zhifa Shen, Ying Tan, Meiyun Zeng, Feng Liu *, and Yuyang Jiang ǁ *, Yingfu Li Department of Chemistry, Tsinghua University, Beijing, , China The Ministry-Province Jointly Constructed Base for State Key Lab- Shenzhen Key Laboratory of Chemical Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong, , China Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main St. W, Hamilton, ON L8S 4K1, Canada Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen, Guangdong, China ǁ Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, , China. address: S-1

2 Scheme S1. SELEX Scheme. The initial DNA library was ligated to the phosphorylated substrate. After incubating with MDA-MB-231 lysate, the catalytically active molecules will cleave the RNA linkage. These molecules will be purified from the uncleaved DNA by 10% dpage. PCR1 was performed to amplify the purified DNA. This is followed by PCR2 using a primer containing an nonamplifiable tag to assist the purification of the desirable DNA strand for the next round of selection. S-2

3 Scheme S2. Recognition of breast tumor target with the RNA-cleaving fluorogenic probe. Conceptual design of the fluorescent probe which can be activate by the target in MDA-MB-231 cell lysate to generate a robust fluorescent signal. S-3

4 Figure S1. Time-dependence reaction of R28 pool incubated with MDA-MB-231 cell lysate. Each sample contained 50 µg/ml protein and 100 nm R28 pool. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. S-4

5 Figure S2. Specificity test of RFD candidates to various breast cell lines. From the top to the bottom are AAI1-1, AAI1-4, AAI2-1, AAI2-4, AAI2-5, AAI2-6 and AAI2-9. Each reaction mixture was analyzed by 10% dpage, followed by fluorimaging. Clv% = cleavage efficiency; NC = negative control. Unclv = uncleaved probe. Clv = cleaved probe. S-5

6 Figure S3. Specificity test of RFD candidates to different cell lines. From the top to the bottom are AAI1-1, AAI1-4, AAI2-1, AAI2-4, AAI2-5, AAI2-6 and AAI2-9. Each reaction mixture was analyzed by 10% dpage, followed by fluorimaging. Clv% = cleavage efficiency; NC = negative control; Unclv = uncleaved probe; Clv = cleaved probe. S-6

7 Figure S4. Western blot of each clinical specimens by detection of β-actin. S-7

8 Figure S5. A 96-well microplate assay of AAI2-5 with clinical specimens. Each sample contained 50 nm probe and 100 µg/ml protein. Incubation time was 30 min. Dark, grey and unfilled circles stand for malignant breast tumor, benign breast tumor, and paracarcinoma or inflammatory tissue, respectively. No. = number of clinical specimens. S-8

9 Figure S6. Cleavage reaction of AAI2-5 with (A) Her-2, ER, PR human recombinant proteins, and (B) serum from breast tumor patients. NC is negative control and MD is MDA-MB-231 cells medium. S-9

10 Figure S7. Flow cytometry assay for the binding of the FAM-labeled sequence AAI2-5-82F (AAI2-5) and R0-82F (R0) with MDA-MB-231 cells (target cells) and MCF-10A cells (control cells). NC = negative control, which was cells incubated with AAI2-5-82F without treatment with fixation/permeabilization reagent. The concentration of the sequences was 100 nm. S-10

11 Figure S8. Fluorescence anisotropy of AAI2-5 incubated with protein above 50 kda from different cell lines. The concentration of probe was 5 nm, and the protein concentration was from 0 to 1300 µg/ml. The measurements were performed in triplicate. The K d values of AAI2-5 for MDA-MB-231 and MCF-10A were determined from a fit of equation mentioned in Experimental Section to these data as ± 8.4 µg/ml, ± µg/ml, respectively. The K d value of AAI2-5 for HEB cannot be determined by this method. S-11

12 Table S1. [Probe] 1/2 value of each probe. Name Selected N40 sequence Repeat times [Probe] 1/2 value AAI1-1 GGGAACGTGTGCTCGGCTATTGATTGGACTTTAGCGTTGG ± 5.1 AAI1-2 CGTAGGCGAAGGTCCGTGTGGGAATCCTTAGCGAGACAAGG ± 8.5 AAI1-3 CATAAATGGTCAGGTGACGTGTACGAAACCTTAGCGTATGG ± 87.8 AAI1-4 GGTCCTGAGAGATACTTTAGCGGTTAGGATACACGAGTGGG ± 3.1 AAI2-1 CAACGAGGGAGCCCTTGGAGAAGTTTAGCGTAGTGGTCGTG ± 2.1 AAI2-3 CACTACGACAGGATTCTGAGTATCGAGTACCTTAGCGTTGC ± 11.4 AAI2-4 GGGTAGTAGTCGAGAGAAGTTTAGCGTAGGCTCGCCGGGTC ± 3.0 AAI2-5 GGAACGGTTAGATCTGATACCTTAGCGAAGGTGTGGTTGGC ± 3.3 AAI2-6 GGATCTGTATCGTGGGAGAATTTTAGCGTCCGAAGCCTGGC ± 5.7 AAI2-7 GTACGGACTCTGAACGATAAGCTTAGCGATCCCGGAAGTGGC ± AAI2-8 GGACGATTGAGAAGTTTAGCGATCAGAAGATGTTGCCGTAGC ± 38.7 AAI2-9 GTGATGTGCTGGCGTATACTTTAGCGTGGATGAGGTCGTGG ± 5.4 AAI2-10 GCCCAAACTTGGAGGGGGGCAATGAGAATCCTTAGCGTGTG ± 11.9 AAI2-11 GGAGACGTGATAGAACTTTAGCGATAAGTAATTTGGATGTGC ± 21.8 AAI2-12 GGGGTACGTTTGATTGACAATCCTTAGCGATATCTGAGTGTG ± 23.3 S-12

13 Table S2 Cleavage efficiency of AAI2-5 and clinical specimens characterization. No. Clv% Source Type Immunohistochemistry cancer breast invasive ductual carcinoma ER (+/-), PR (+), Her-2 (+/-), CK5/6 (-), Ki-67 (+) 7% cancer breast invasive ductual carcinoma ER (++), PR (+++), Her-2 (+/++), Ki-67 (+) 40%-50% cancer breast invasive ductual carcinoma ER (+/-), PR (+/-), Ki-67 30% paracarcinoma breast invasive ductual carcinoma ER (++), PR (-), Her-2 (+++), CK5/6 (+), Ki-67 (+) 30%-40% paracarcinoma breast invasive ductual carcinoma ER (+), PR (+), Her-2 (-) cancer breast invasive ductual carcinoma;grade II ER (-), PR (-), Her-2 (+++), Ki % cancer breast invasive ductual carcinoma;grade II ER (+) 20%, PR (-), Her-2 (++),, P53 (+), Ki-67 (+) 20% cancer intracystic papillary carcinoma ER (+) 90%, PR (+) 70%, Her-2 (-), Ki-67 (+) 5% paracarcinoma breast invasive ductual carcinoma ER (++), PR (+), Her-2 (+), Ki-67 (+) 15% tumor breast fibroadenoma lymph breast invasive ductual carcinoma;grade II ER (+++), PR (++), Her-2 (+/++) tumor intraductal papilloma granulomatous lobular mastitis cancer breast invasive ductual carcinoma;grade II ER (-), PR(-), Her-2 (+++), Ki-67 15%, cancer breast invasive ductual carcinoma;grade II ER (+/++), PR (-), Her-2 (+++), Ki-67 5% granulomatous lobular mastitis cancer breast invasive ductual carcinoma;grade II ER (+), PR (-), Her-2 (+++), Ki-67 9%, cancer breast invasive ductual carcinoma;grade II ER (++) 60%, PR (++) 70%, Her-2 (+++), P53 (-), Ki-67< 10% (+) cancer breast invasive ductual carcinoma;grade III ER (-), PR (-), Her-2 (+++), P53 (-), Ki-67 (+) 30% tumor phyllodes tumor tumor breast fibroadenoma paracarcinoma breast invasive ductual carcinoma;grade II ER (+++), PR (++), Her-2 (+/++) paracarcinoma breast invasive ductual carcinoma;grade II ER (-), PR (-), Her-2 (+++), Ki % cancer breast invasive ductual carcinoma;grade II ER (+++), PR (+++), Her-2 (-), Ki-67 5% tumor breast fibroadenoma paracarcinoma breast invasive ductual carcinoma ER (++), PR (-), Her-2 (++/+++), Ki-67 67% paracarcinoma breast invasive ductual carcinoma ER (+/-), PR (+), Her-2 (+/-), CK5/6 (-), Ki-67 (+) 7% tumor breast fibroadenoma tumor breast fibroadenoma cancer breast invasive ductual carcinoma;grade III ER (+) 90%, PR (-), Her-2 (-), P53(+), ki-67 (+) 40% granulomatous lobular mastitis tumor phyllode tumor cancer breast invasive ductual carcinoma;grade II ER (+) 70%, PR (-), Her-2 (+/-), Ki-67 (+) 20%, P53 (+) cancer infiltrative breast cancer with neuroendocrine differentiation ER (+) 60%, PR (-), Her-2 (-), Ki-67 (+) 10%, P53 (-), NSE (+), syn (+) fibroadenosis paracarcinoma breast invasive ductual carcinoma ER (++), PR (++), Her-2 (++), Ki-67 (+) 20% tumor breast fibroadenoma cancer breast invasive ductual carcinoma;grade II ER (-), PR (-), Her-2 (+++), Ki % tumor breast fibroadenoma paracarcinoma breast invasive ductual carcinoma ER (-), PR (-), Her-2 (+++), CK5/6 (+), Ki-67 35% cancer breast invasive ductual carcinoma;grade II-III ER (-), PR (-), Her-2 (+), Ki-67 (+) 50% tumor intraductal papilloma cancer breast invasive ductual carcinoma; ER (+) 50%, PR (+) 30%, Her-2 (++), Ki-67 (+) 60%, P53(+) breast invasive ductual carcinoma;grade II ER (-), PR (-), Her-2 (+++), P53 (-), Ki-67 (+) 20% tumor phyllodes tumor paracarcinoma breast invasive ductual carcinoma;grade II ER (+), PR (-), Her-2 (+++), Ki-67 9% cancer breast invasive ductual carcinoma;grade II ER (++) 70%, PR (-), Her-2 (-), Ki-67 (+) 20%, P53 (-) sclerosing adenosis cancer breast invasive ductual carcinoma;grade III ER (+) 70%, PR (+) 2%, Her-2 (++), Ki-67 (+) 40%, P53 (+) cancer intracystic papillary carcinoma ER (+) 80%, PR (+), 30%, Her-2 (-), Ki-67 (+) 20% granulomatous lobular mastitis cancer breast invasive ductual carcinoma;grade III ER (-), PR (-), Her-2 (+++), P53 (+), Ki-67 (+) 20% tumor breast fibroadenoma tumor adenosis with breast fibroadenoma S-13