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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, Yuyang Jiang, and Yingfu Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5031557 • Publication Date (Web): 05 Dec 2014 Downloaded from http://pubs.acs.org on December 8, 2014

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Analytical Chemistry

Highly specific recognition of breast tumors by an RNAcleaving fluorogenic DNAzyme probe Shengnan He † ⊥ ‡, Long Qu † ⊥ § ‡, Zhifa Shen §, Ying Tan ⊥, Meiyun Zeng ▽, Feng Liu ⊥ *, Yuyang Jiang † ⊥ ǁ *, and Yingfu Li§ †

Department of Chemistry, Tsinghua University , Beijing, 100084, 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, 518055, 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, 100084, China. ABSTRACT: Breast cancer is one of the most commonly diagnosed cancers among females worldwide. Early detection of breast cancer is of vital importance to the reduction of the mortality rate. However, the lack of specific biomarkers that can effectively identify breast cancer cells limits the ability for early diagnosis of breast cancer. RNA-cleaving fluorogenic DNAzymes (RFDs), which can be produced through the Systematic Evolution of Ligands by Exponential enrichment (SELEX) process, are catalytic DNA molecules capable of generating a fluorescent signal when the appropriate target is bound. In this study, we carried out a SELEX experiment to select for RFDs that are active in the cell lysate of MDA-MB-231, a model breast cancer cell line. We obtained a RFD probe, named AAI2-5, that can detect MDA-MB-231 at a concentration of cell lysate proteins as low as 0.5 µg/mL (which is equivalent to ~5,000 cell/mL). AAI2-5 is capable of distinguishing MDA-MB-231 cells from normal cells as well as other types of tumor cells, including other subtypes of breast cancer cells. Moreover, AAI2-5 responded positively to more than 90% of malignant breast tumors. This report is the first study to explore the RFD system for the detection of cancer cells. The results suggest that RFD can be potentially applied for the diagnosis and treatment of breast cancer in the future.

Breast cancer is the most frequently diagnosed cancer in females and ranks second among all cancers when both genders are considered. Approximately 1.3 million breast cancer cases are estimated to occur each year worldwide, leading to 14% of all cancer-related deaths.1 The incidence of breast cancer has increased over the past decade and is expected to rise substantially in the coming years.2 Therefore, breast cancer will remain a significant human health problem and a huge burden on healthcare systems. The importance of early detection for the control of the mortality rate associated with breast cancer has been well demonstrated. It was found that in Sweden, the mortality of women with breast cancer decreased by 63% during the period of 1988-1996 compared to the period between 1968-1977. This finding was attributed to the availability of mammography screening for early detection of breast cancer.3 The two most widely used methods for early screening of breast cancer are mammography and magnetic resonance imaging (MRI), both of which are very costly, and require special equipment as well as highly trained tehcnicians.4-6 In addition, these techniques lack accuracy and sensitivity.7 Therefore, new and simpler methods are needed to better detect breast cancer cells in patients. The development of more

cost-effective and convenient tests for early diagnosis of breast cancer that could be adopted both in developed countries and developing countries is of the utmost urgency. In recent years, functional nucleic acids such as aptamers and DNAzymes (also called deoxyribozymes) have been used as effective molecular tools to detect specific targets as well as discover new biomarkers of diseases.8, 9 Both aptamers and DNAzymes are usually generated via Systematic Evolution of Ligands by Exponential enrichment (SELEX).10-13 Many DNA and RNA aptamers have been derived to recognize a diverse range of targets including proteins, lipids, saccharides, nucleotides, metal ions, with high specificity and affinity.14-23 DNA aptamers have also been selected to specifically recognize a host of tumor cells, such as human Burkitt’s lymphoma cells,24 glioblastoma cells,25 liver cancer cells,26 metastatic breast cancer cells,27 acute leukemia cells,28 brain tumor-initiating cells.9 DNAzymes were first discovered in 1990s.29 Since then many DNAzymes have been reported for catalysing many different chemical transformations.30-39 Some DNAzymes have also been used for the development of biosensors.40-43 For example, DNAzymes that bind to specific metal ions have been developed and used to detect toxic metal ions such as Pb (II),44 Cu (II),45, 46 Hg (II).47 For the past 10 years, Li and colleagues have developed a biosensing platform named “RNA-cleavage fluorogenic

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DNAzyme (RFD)”. This system is based on both aptamer and DNAzyme technologies.44, 48-50 The RFD probe is a DNA chain with one single RNA linkage inserted. The RNA unit is flanked by nucleotides labelled with a fluorophore and a quencher. An RFD can fold into an RNA-cleaving conformation after binding with a specific target, resulting in the separation of the fluorophore from the quencher and generation of a fluorescent signal. As reported previously, in vitro selection has been successfully applied to the selection of RFDs that can detect Escherichia coli (E. coli) cells. The selected E. coli- sensing RFD shows excellent detection sensitivity and specificity.51, 52 Furthermore, the RFD probe can be used to set up a mix-and-read type of assay which is easy to perform. At the same time, conventional approaches for the development of disease-specific molecular probes take a massive amount of time and involve complex steps for identification of disease biomarkers. In contrast, the RFD selection process requires no prior knowledge of targets. For these reasons, we set out to investigate whether it was possible to develop effective RFD probes for breast tumor detection. In this study, we have isolated an RFD probe that can be used to achieve specific and sensitive detection of the breast cancer cell line MDA-MB-231. We have also found that the RFD exhibits a good degree of specificity in recognizing clinical tumor specimens. Experimental Section Materials and specially prepared reagents T4 DNA ligase, T4 polynucleotide kinase (PNK), Taq DNA polymerase, dNTPs, and ATP were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). Ampicillin, N,N,N,N’-tetramethylethylenediamine (TEMED), and 40% solution of acrylamide/bis-acrylamide (29:1) mixture was obtained from Sangon Biotech (Shanghai, China). pGEM-T Vector Systems I was obtained from Promega (USA). It contains a TA cloning vector (pGEM-T), 2× rapid ligation buffer, T4 DNA ligase. Complete Protease Inhibitor Cocktail Tablets were obtained from Roche Ltd. (China). The cytoplasmic/nuclear protein extraction kits and BCA protein assay kits were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The fixation/permeabilization reagent for flow cytometry was obtained from ebioscence (USA). L-15 medium was obtained from Gibco (USA). Dulbecco’s modified Eagle’s medium (DMEM), Iscove’s Modified Dubecco’s Medium (IMDM), RPMI-1640, and fetal bovine serum (FBS) was obtained from Hyclone (USA). All other chemicals were purchased from Sigma-Aldrich (USA) and used without further purification. E. coli JM109 is routinely maintained in our laboratory. Human epidermal growth factor receptor 2 (HER2) full-length ORF recombinant protein (a.a.1-1255) with a GST tag at the N-terminus was obtained from Abnova Corporation (Taipei, Taiwan). The progesterone receptor (PR) human recombinant protein (a.a.412-562) with GST tag and estrogen receptor (ER)-alpha human recombinant protein (a.a.1-300) were obtained from ProSpec Tany TechnoGene Ltd (Israel). 0.22 µM filter was from Merck Millipore (Germany) and nanosep centrifugal devices with Omega membranes were from Pall Corporation (USA). All clinical specimens were obtained either from the Second People’s Hospital of Shenzhen (Shenzhen, China) or Peking University Shenzhen Hospital (Shenzhen, China). The water used in this study was doubledeionized (ddH2O) and autoclaved.

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2× selection buffer contained 100 mM HEPES (pH 7.5), 400 mM NaCl, and 10 mM MgCl2. Lysis buffer contained 10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 0.1% NP40, and 1× complete Protease Inhibitor Cocktail. All solutions were freshly prepared before use. Cell lines and cell culture All cell lines were obtained from the cell bank, Shanghai Institutes for Biological Sciences. The MDA-MB-231 (mammary gland adenocarcinoma) cell line was cultured in L-15 medium, supplemented with 10% FBS; 37 ºC; air 100%. The ZR-75-1 (mammary gland ductal carcinoma), SK-BR-3 (mammary gland adenocarcinoma), BT-474 (mammary gland ductal carcinoma) BT-549 (mammary gland ductal carcinoma), MCF 10A (mammary gland fibrocystic disease), Hs 578Bst (mammary gland normal), BEL-7404 (hepatocellular carcinoma), HeLa (cervical adenocarcinoma), CCRF-CEM (acute lymphoblastic leukemia), K562 (chronic myelogenous leukemia), NCI-H69 (small cell lung cancer), HL-7702 (normal liver), Chang liver (normal liver), and QSG-7701 (normal liver) cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS. The MCF-7 (mammary gland adenocarcinoma), Hep G2 (hepatocellular carcinoma), SK-HEP-1 (liver adenocarcinoma), A172 (glioblastoma), U251 (glioblastoma), HEB (normal brain), and 293T (human embryonic kidney) cell lines were cultured in DMEM supplemented with 10% FBS. HCT 116 (colorectal carcinoma) cell line was cultured in IMDM supplemented with 10% FBS. NCM460 (normal transverse colon) cell line was cultured in Ham’s F-12 supplement with 10% FBS. All cells were cultured at 37 ºC in a humidified atmosphere of 5% CO2 and 95% air except for MDA-MB231 cells. Sample preparation Cells were washed twice with PBS and scraped into cell lysis buffer. Cell suspensions were sonicated for 4 s and 9 s intervals for 3 cycles. Then they were put on ice for 30 min, inverting the tube every 5 min. The cell lysate was centrifuged at 20,000 g, 4 ºC for 15 min. Supernatant was collected for use. For clinical specimens, solid tumors or other tissues were minced and ground in a homogenizer. Both tumor and tissue homogenates were sonicated for 5 s, rested for 10 s, and this was repeated for 30 cycles. The sonicated cell homogenate was then centrifuged at 20,000 g, 4 ºC for 15 min. Supernatants were collected for use. Blood from breast tumor patients was rested at room temperature for 30 min. Then it was centrifuged at 2000 rpm, room temperature for 20 min. Serum was collected and filtered with a 0.22 µM filter. All patients were informed and they signed consent forms to provide samples for the experiments. For target protein identification, MDAMB-231 cell proteins were separated using a cytoplasmic/nuclear protein extraction kit. For the molecule weight assessment of the target, proteins were separated by Nanosep centrifugal devices with Omega membranes. Sequences of Oligonucleotides A single strand DNA library which contains 40 nucleotides (nt) in the middle of the sequence was used for SELEX. The sequence, DL1, is: 5’-CACGG ATCCT GACAA G-N40CAGCT CCGTC CG-3’, N40 = 40 random nucleotides sequence (N = an equimolar mixture of A, C, G and T). The primers FP (5’-CACGG ATCCT GACAA G-3’) and RP1 (5’CGGAC GGAGC TG-3’) were used in PCR1; FP and RP2 (5’-A20-S9-CGGAC GGAGC TG-3’, S9 is a triethylene gly-

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Analytical Chemistry

col linker, "Spacer 9") were used in PCR2. Template (5’CTAGG AAGAG TCGGA CGGAG CTG-3’) and substrate (5’-ACTCT TCCTA GCFRQ GGTTC GATCA AGA-3’, F, R and Q represent a fluorescein-labeled dT, an adenine ribonucleotide, and a DABCYL-labeled dT, respectively) were used for ligation. The sequence of AAI2-5-82F employed for flow cytometry and fluorescence anisotropy is as follows: 5’CACGG ATCCT GACAA GGGAA CGGTT AGATC TGATA CCTTA GCGAA GGTGT GGTTG GCCAG CTCCG TCCGA

CTCTT CCTAG CF-3’, and F represents a FAM-labeled dT. The control sequence R0-82F is as follows: 5’-CACGG ATCCT GACAA G-N40-CAGCT CCGTC CGACT CTTCC TAGCF-3’, N40 = 40 random nucleotides (N = an equimolar mixture of A, C, G and T), and F represents a FAM-labeled dT. All oligonucleotides were synthesized by Takara Biotechnology Co., Ltd. (Dalian, China). SELEX protocol The SELEX scheme is shown in Scheme S1. The substrate was phosphorylated and ligated with DL1 in the presence of template. The ligated product was purified by 10% denaturing polyacrylamide gel electrophoresis (dPAGE), and incubated with MDA-MB-231 cell lysate for 2 h at room temperature. 10% dPAGE was used to separate the cleaved DNA from uncleaved DNA. The process of PCR1 began with denaturation at 94 ºC for 1 min, 12-17 cycles of amplification (94 ºC for 30 s, 56 ºC for 45 s, 72 ºC for 45 s) and finally a single extension step of 72 ºC for 1 min. The PCR2 conditions employed were the same as those indicated for PCR1. PCR2 products were separated by 10% dPAGE and ligated to the phosphorylated substrate for the next round of SELEX. To evaluate the selection procedure, the cleavage efficiency (Clv%) was calculated as follows: Clv% = (Int[clv] / 6) / (Int[clv] / 6 + Int[unclv] ) × 100% Int[clv] is the intensity of cleaved DNA band and Int[unclv] is the intensity of the uncleaved DNA band. To evolve DNAzymes with high affinity and specificity, the incubation time was reduced from 2 h to 30 min and the amount of cell lysate used was reduced from 50 µg to 10 µg, and counter-SELEX was used to decrease the non-specific binding. The detailed procedure used for DNAzyme selection has been published.53 Sequencing of the selected DNAs After 28 rounds of selection, DNA pools were amplified by PCR: denaturation at 94 ºC for 1 min, 12-17 cycles of amplification (94 ºC for 30 s, 56 ºC for 45 s, 72 ºC for 45 s) and finally a single extension step of 72 ºC for 10 min. PCR products were ligated with T-vector at 4 ºC overnight. Ligation fragments were transformed into E coli JM109 competent cells. Positive clones were selected and sequenced by Invitrogen (Guangzhou, China). Activity assay of the selected probes The DNAzyme activity assay was carried out by measuring fluorescence intensity. [Probe]1/2 (P1/2) was used to estimate the activity of probe candidates. It is defined as the concentration of the AAI2-5 probe causing half of the maximal fluorescence in the presence of a given amount MDA-MB-231 cell lysate.10 mg/L of MDA-MB-231 cell lysate was mixed with various concentrations of probes (0-680 nM) in selection buffer. The fluorescence intensity was measured on a DTX 880 multimode detector (Beckman Coulter, USA) after incubation at 25 ºC for 20 min. The emission signal (λmax = 525

nm) was recorded upon excitation at 470 nm. Reactions were performed in triplicate. The experimental data were fitted to the following equation: Fob = Fmax [probe]/ (P1/2+[probe]) The observed relative fluorescence intensity (Fob) for each probe concentration was calculated using the equation Fob = F F0 where F is the fluorescence intensity at the given probe concentration, F0 is the intensity at 0 nM probe concentration, Fmax is the value of the Fob with excessive amount of probe.. Sensitivity of AAI2-5 MDA-MB-231 cell lysate was prepared and quantified using a BCA kit. Cell lysate was diluted into a series of concentrations with lysis buffer. Each cleavage reaction was set up as follows: 0.2 mM EGTA, 90 µL of lysate dilution, 100 µL of 2× selection buffer, 50 µM AAI2-5, and ddH2O was added to a final volume of 200 µL. The fluorescence signal was monitored by a Horiba Jobin Yvon Spectrophotometer (excitation wavelength = 488 nm and emission wavelength = 517 nm) at room temperature. The fluorescence intensity was recorded every 20 s for 20 min. Following this step, the solution of each group was precipitated with ethanol. The cleavage reactions were analysed by 10% dPAGE. The experiment was performed in triplicate. The sensitivity of AAI2-5 was determined to be the lowest concentration of cell lysate that caused substrate cleavage. Fluorescence anisotropy measurements All fluorescence anisotropy measurements were carried out at 25 ºC in 96-well assay plate (Costar, USA) using microplate reader (Tecan infinite M1000 Pro, Switzerland). Measurements were performed by recording the fluorescence emission intensities at 520 nm (5 nm bandwidth) using excitation at 470 nm (5 nm bandwidth). 5 nM of AAI-2-5-82F was incubated with a series of concentrations of cell lysate protein above 50 kDa (0-1300 µg/mL) from MDA-MB-231, MCF-10A, or HEB for 20 min. The measurements were conducted in triplicate. The experimental data were fitted to the following equation: △r = Bmax [pro] / (Kd + [pro]) where △r is the anisotropy change from free probe to interaction with the given concentration of MDA-MB-231 lysate protein [pro], Kd is the dissociation constant, and Bmax is the difference in fluorescence anisotropy between free and fully bound AAI2-5-82F complex. Flow cytometry Flow cytometry was carried out to locate the target of AAI2-5. 1× 106 of MDA-MB-231 or MCF-10A cells were treated with fixation/permeabilization reagent (ebioscience, USA) previously and incubated with FAM-labeled probe AAI2-5-82F at the final concentration of 100 nM in 1× selection buffer at room temperature for 30 min. Cells were washed twice with wash buffer to remove the unspecific binding and resuspended in 100 µL of wash buffer. The fluorescence intensity was recorded by a MoFlo highperformance cell sorter (Beckman counter, USA) by counting 10000 events. Cells without incubation and the FAM-labeled initial ssDNA library R0-82F were used as negative controls. Results and Discussion SELEX of breast cancer specific probes The SELEX strategy used in this work is illustrated in Scheme S1, and the detailed procedures are provided in the

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experimental section. There were two main reasons that MDA-MB-231 cell lysates were directly used as the complex target of our RFD probe selection. First of all, the liganddependent RFD probe is a combination of an aptamer for specific binding to a given target and a DNAzyme for RNA cleavage, which is triggered by aptamer-ligand binding. Thus, selecting such a DNAzyme with both binding and catalytic functions is viewed to be more challenging than selecting an aptamer alone. Secondly, cell lysate contains a large number of potential targets including proteins and metabolites. Therefore, as a mixture, cell lysate would offer a better chance for successful selection of a RFD probe. Use of cell lysate for RFD selection can also avoid the steps for identification of specific targets which are usually time-consuming and costly. Previous work however has already proven that it is feasible to select RFD probes without knowing and purifying specific targets. The fluorescent probe is designed to cleave an RNA linkage upon contacting the target cell lysate. The cleavage site is located between two nucleotides labeled with a fluorophore and a quencher, respectively. The cleavage event therefore, will separate the quencher from the fluorophore, resulting in the generation of a robust fluorescent signal (Scheme S2). The progress of selection was monitored by 10% dPAGE. An observable fluorescent signal was generated starting at the 4th round of selection. To derive DNA probes with high affinity, the incubation time was shortened from 2 h to 30 min, and the amount of MDA-MB-231 cell lysate used in the selection was decreased from 500 µg/mL to 100 µg/mL. To ensure recognition specificity, the lysate from the normal breast cell line MCF-10A was used in a counter-selection step. After 28 rounds of selection, RFD candidates responding to the target cell lysate were enriched, as judged by the percentage of cleavage (Clv%, calculated as described in the experimental section). As shown in Figure 1A, cleavage activity was increased progressively from round 1 to round 28. Furthermore, the Clv% values at round 25 (R25) and round 28 (R28) were approximately equal (R25 = 25.9%, R28 = 27.7%), which indicated that 28 rounds of selection were adequate in enriching a pool of DNA sequences that efficiently responded to MDA-MB-231 cell lysate. Figure S1 shows the timedependent reaction of the R28 DNA pool with MDA-MB-231 cell lysate. As the reaction time was extended, the fluorescent signal increased. Analysis of the R28 DNA pool revealed significant specificity to MDA-MB-231 cell lysate as compared with other different breast cell line lysates (Figure 1B), as well as other tumor and normal cell line lysates (Figure 1C). Considering the stable cleavage percentage as well as excellent selectivity, the R28 pool was used for cloning and sequencing to find suitable probes. Identification of probe candidates After 28 rounds of the enrichment, the selected DNA pool was PCR-amplified, ligated with T-vector and transformed into E. coli JM109. Positive clones were sequenced. Analysis of the sequencing results indicates that the selected RFD candidates can be categorized into 15 families based on their sequence homology. 15 different DNA molecules, one from each family, were taken as probe candidates and synthesized. In the first step, we determined the P1/2 value of each RFD candidate, defined as the probe concentration that causes half of the max-

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imal fluorescence in the presence of a given amount MDAMB-231 cell lysate, as a way to compare their relative efficiency in recognizing MBA-MB-231. Most of the selected probes had a P1/2 in the nanomolar range (Table S1, Figure 2A). Next, to further characterize the sequences, breast cell lines and other cell lines were used to test the specificity of the RFD candidates with a P1/2 value less than 50 nanomolar (Figure S2, S3). The Clv% of each sequence towards different cell lines was compared with the value of MDA-MB-231, since all the probes showed a high degree of specificity for this cell line. AAI2-5 was chosen for further studies due to its better specificity (Figure 2B and C, Table 1 and 2) and higher binding ability (Figure 2A). Interestingly, AAI2-5 was able to distinguish MDA-MB-231 from other subtypes of breast cancer cells (Figure 2B, Table 2). Although AAI2-5 had a weak response towards MCF-10A, the Clv% value for MCF-10A was much smaller than that observed for MDA-MB-231. Meanwhile, this probe exhibited weak responses towards cervical cancer, glioblastoma, leukemia, colon cancer, liver cancer, and other normal cell lines (Figure 2C, Table 2). Taken together, the data suggest that AAI2-5 is quite specific for MDA-MB231.

Figure 1. In vitro selection results and specificity of R28 DNA pool. (A) RNA-cleaving activity of different DNA pools in the presence of MDA-MB-231 cell lysate. Marker represents the expected cleavage product, which can be observed by fluorescence scan as it contains the fluorophore. Each DNA pool was incubated with 100 µg/mL MDA-MB-231 cell lysate for 30 min. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. (B) Activity of Round 28 pool towards different breast cell lysates. Hs 578Bst and MCF-10A are normal breast cell lines and others are tumor cell lines. (C) Activity of Round 28 pool towards lysates of non-breast cell lines. HL-7702 is a normal cell line and others are tumor cell lines (see Materials and methods for details).

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Analytical Chemistry

Table 1. Specificity test of representative probes to various breast cell linesa.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MDA-MB-231

ZR-75-1

SK-BR-3

BT-474

MCF-7

BT-549

Hs 578Bst

MCF-10A

AAI1-1

++++

++

++

++

++

++

+++

++

AAI1-4

++++

++

++

++

+++

++

++

+++

AAI2-1

++++

+++

++

+

++

++

+++

+++

AAI2-4

++++

+++

+

+

+

+

++

+++

AAI2-5

++++

+

+

+

+

+

+

++

AAI2-6

++++

+++

++

+

+

++++

+++

+++

AAI2-9

++++

+++

++

+

+

+++

+++

+++

a The cleavage efficiency of each RFD candidate towards different breast cell lines was compared with MDA-MB-231. The percentage of probe cleavage was used to evaluate the specificity of these sequences: 85%: ++++. The final concentration of each sequence in reaction buffer is 50 nM.

Table 2. Specificity test of representative probes to different cell linesa. breast

liver

cervical

glioblastoma

leukemia

colon

lung

normal

MDAMB-231

BEL7404

Hep G2

SKHEP-1

Hela

A172

U251

CCRFCEM

K562

HCT 116

NCIH69

HL-7702

AAI1-1

++++

+++

+++

+

++

+++

+++

++

+++

+++

+++

+++

AAI1-4

++++

+++

-

-

-

+++

+++

++

++

++

+

++

AAI2-1

++++

+++

++

++

++

++

+++

++

++

++

+

++

AAI2-4

++++

++

++

++

+

++

+

++

+

+

+

++

AAI2-5

++++

++

++

++

+

+

+

+

+

+

+

+++

AAI2-6

++++

+++

++

+

+

+++

++

++

+

++

+++

+++

AAI2-9

++++

+++

+++

++

++

++++

+++

+++

+++

++

++

++

a

The cleavage efficiency of each RFD candidate towards different cell lines was compared with MDA-MB-231. The percentage of probe cleavage was used to evaluate the specificity of these sequences: 85%: ++++. The final concentration of each sequence in reaction buffer is 50 nM.

The sensitivity of AAI2-5 was also evaluated by monitoring the real-time fluorescence change. According to the results in Figure 3, the fluorescence signal of AAI2-5 rised as the protein concentration increased from 0.5-1000 µg/mL. The reaction mixtures were also analyzed by 10% dPAGE and 96well microplate image (Figure 3B). Both experiments showed the probe could detect as low as 0.5 µg/mL of protein. This concentration of protein translates to 4808 cell/mL. Taken together, AAI2-5 can recognize MDA-MB-231 with great specificity and sensitivity. Specific recognition of breast tumor specimens To test if AAI2-5 can recognize clinical tumor specimens, we examined 54 patient samples obtained from two local hospitals (see Material and Methods). Sample protein was quantified using a BCA protein assay kit, and the levels of protein in each sample were confirmed by blotting for β-actin protein

(Figure S4). Among the 54 breast specimens, there were 24 malignant tumor samples, 13 benign tumor samples, and 17 paracarcinoma or inflammatory tissue samples. The detailed information, including the source, disease type, and immunohistochemistry of each sample, is listed in Table S2. AAI2-5 was incubated with different samples separately. The results are shown in Figure 4 and Figure S5. AAI2-5 displayed significant cleavage with 36 specimens (Clv% above 10%, Figure 4A, Table 3), most of which were tumors (34 tumors and 2 non-tumors). The two non-tumor samples triggered only a low level of cleavage activity with Clv% of 11% and 13%. These results suggest that AAI2-5 can recognize breast tumors regardless of whether they are benign or malignant. The Clv% for clinical specimens varied, suggesting that the targets of this probe might be expressed differently in these samples (Figure 4A, Figure S5, and Table 3). Examination of the statistical data in Figure 4B reveals that this probe can discriminate a

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receptors for the definition of breast cancer subtypes in clinical practice. Therefore, we wondered if the cleavage ability of AAI2-5 was associated with expression level of these three proteins. Figure S6A showed that AAI2-5 had no cleavage activity in response to the recombinant proteins of HER2, PR, and ER. This result is in accordance with the triple negative receptor phenotype of the target cell line MDA-MB-231, since MDA-MB-231 does not express the genes for these three receptors. In addition, the probe can specifically identify triple negative breast cancer (TNBC)-type cell line MDA-MB-231 from other breast cancer cell line subtypes, but failed to distinguish HER2, PR, or ER negative expression specimens from positive expression ones. This result might be due to a difference in expression of a particular molecule between cell lines and clinical specimens. In addition, the cleavage activity of AAI2-5 was tested with the blood serum from breast tumor patients. The negative results presented in Figure S6B indicate that the targets of the probe were not secretory products of breast tumors.

Figure 2. Characterization of AAI2-5. (A) P1/2 of AAI2-5 showed its catalytic activity with MDA-MB-231 cell lysate. The data was the average of three independent experiments. (B) AAI2-5 was incubated with 50 µg/mL of different breast cell lines lysate for 20 min. MCF-10A and Hs 578Bst are normal breast cells, while others are breast cancer cells. C, AAI2-5 was incubated with 50 µg/mL of different cell lines lysate for 20 min. Chang Liver, HL7702 and QSG 7701 are normal human liver cell lines; HEB is normal human brain cell line; NCM460 is transverse colon cell line; 293T is human embryonic kidney cell line; others are cancer cell lines. NC was negative control, which was AAI2-5 incubated with 2× selection buffer plus lysis buffer for 20 min. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe.

tumor from normal tissue based on the cleavage efficiency. The results strongly suggest that the target of AAI2-5 might be a good indicator of breast tumors. In addition, the excellent specificity of AAI2-5 makes it a promising tool for the identification and characterization of new biomarkers that may be used to simplify clinical diagnosis of breast tumors. Human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and estrogen receptor (ER) are three

Figure 3. Sensitivity of AAI2-5 towards MDA-MB-231 cell lysate. (A) Real-time fluorescence monitoring over the background fluorescence. Protein concentration was considered for detection limits. (B) Rate of signal increase in response to the protein concentration in MDA-MB-231 cell lysate. Inset shows the linear response at low protein concentrations. RF = Relative Fluorescence. The data is the average of three independent experiments. (C) 10% dPAGE and the corresponding 96-well microplate image of the probe incubated with MDA-MB-231 cell lysate (0-1000 µg/mL protein) for 60 min. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe.

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Analytical Chemistry

Figure 4. Reactivity of AAI2-5 towards clinical specimens. (A) 10% dPAGE analysis of AAI2-5 incubated with clinical specimens. Each sample contained 50 nM probe and 100 µg/mL protein. Incubation time was 30 min for each group. Dark, grey and unfilled circles represent malignant breast tumor, benign breast tumor, paracarcinoma (or inflammatory tissue), respectively. No. = number of clinical specimens; Clv % = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. (B) Statistics of data in Figure 4A.

Table 3. Cleavage efficiency of AAI2-5 incubated with clinical specimens. Tumor Clv%

Malignant

Benign

N = 24

N = 13

2

1

15

10-35 %

9

6

2

35-60 %

8

5

0

60-85 %

2

1

0

> 85%

3

0

0

< 10% > 10%

Normal

The core sequence identification of AAI2-5 The sequence “TTAGCG” was found in all clones that were subjected to sequencing analysis. To investigate the importance of this sequence, a mutant sequence (AAI2-5-M) having “TTTTTT” instead of “TTAGCG” and a deletion sequence (AAI2-5-D) with the “TTAGCG” deletion were employed in cleavage assays. Also, every nucleotide of “TTAGCG” was mutated to examine the key nucleotides for interaction with MDA-MB-231. The activity analysis of all these sequences are shown in Figure 5. For this experiment, 50 µg/mL of MDA-MB-231 cell lysate was incubated with 25 nM of each ssDNA for 6 h. There was a large fluorescent signal generated when AAI2-5 was used in the cleavage reaction, whereas AAI2-5-M and AAI2-5-D showed little fluorescent signal change when mixed with the MDA-MB-231 cell lysate (Figure 5B and 5C). Meanwhile, the fluorescence changes of single-nucleotide mutants were larger than AAI2-5-M and AAI2-5-D, but much smaller than the original probe. Taking together, these results indicate that the “TTAGCG” sequence is necessary either for the enzymatic or the binding activity of AAI2-5.

N = 17

Total

Tumor: 8.1% (3/37) Normal: 88.2% (15/17) Tumor: 91.9% (34/37)

Normal: 11.8% (2/17)

Target characterization of AAI2-5 The target that activates AAI2-5 could be a variety of molecules including proteins and small-molecule metabolites. Firstly, we tested whether the target of AAI2-5 was a protein. For this experiment, the concentration of cell lysates was quantified by using a BCA protein assay kit. 5 µg protein of MDA-MB-231 cell lysate was treated either by proteinase K at 55 ºC for 2 min, or by trypsin at 37 ºC for 2 min. Each digested sample was then incubated with 10 pmol of AAI2-5 for 75 min. Figure 6 shows that AAI2-5 was very active with undigested cell lysate but was inactive when incubated with either of the digested samples. The results indicated that the targets of AAI2-5 were most likely proteins. Flow cytometry was performed to identify whether the target is located on the membrane of MDA-MB-231 (Figure S7). The negative control group (NC) was incubated with FAM-labeled AAI2-5 without the treatment with the fixation/permeabilization reagent, which maintained cell integrity. Compared with NC, the group incubated with FAM-labeled AAI2-5 with the treatment showed significantly higher fluorescence. This experiment

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5-fold smaller than that from MCF-10A. The signal obtained with the HEB derived protein is too weak to allow the calculation of apparent Kd. The results from this experiment are consistent with the early observation that AAI2-5 has a strong selectivity for MBA-MB-231. To determine whether the targets of AAI2-5 were secreted proteins, serum derived from patients’ blood as well as conditioned medium from MDA-MB-231 cells were tested to see whether they could trigger the fluorescent signal generation. The results show that neither patient serum nor MDA-MB-231 conditioned medium was successful in generating a fluorescent signal (Figure S6B). These results suggested that the targets of AAI2-5 are not produced by extracellular secretion. Based on our preliminary experiments for target identification, the target for AAI2-5 most probably is a cytoplasmic protein with a molecular weight greater than 50 kDa. Future studies will utilize bioanalytical techniques to identify the target of AAI2-5 in MDA-MB-231 cells. However, the final identification of the AAI2-5 target will be a long-term, multistep process, which is beyond the scope of this study and will be pursued and presented in our future reports.

Figure 5. The sequence “TTAGCG” of AAI2-5 is important for its cleavage ability. (A) Mutant sequences of AAI2-5. The sequence motif “TTAGCG” of AAI2-5 was mutated into “TTTTTT” (AAI2-5-M), or deleted (AAI2-5-D). Each nucleotide of the motif was also mutated (AAI2-5-1, AAI2-5-2, AAI2-5-3, AAI2-5-4, AAI2-5-5 and AAI2-5-6). △ = the motif deleted; F, R and Q represent fluorescein-labeled dT, adenine ribonucleotide, and DABCYL-labeled dT, respectively. (B) Time-dependent fluorescence change of AAI2-5, the deletion sequence (AAI2-5-D) and mutated sequences (AAI2-5-1, AAI2-5-2, AAI2-5-3, AAI2-5-4, AAI2-5-5, AAI2-5-6, AAI2-5-M) incubated with 50 µg/mL of MDA-MB-231 cell lysate. The probe concentration was 25 nM. RF = Relative Fluorescence. (C) 10% dPAGE results of the sequences in panel A incubated with 50 µg/mL of MDA-MB-231 cell lysate for 6 h. NC = negative control; Unclv = uncleaved probe; Clv = cleaved probe.

suggests that the target of AAI2-5 was not located on the cell membrane. Next, MDA-MB-231 cell lysate was separated into nuclear and cytoplasmic protein fractions. As shown in Figure 6C, AAI2-5 had similar cleaving activity with cytoplasmic and whole protein fractions, whereas it was inactive with proteins present in the nuclear fraction. The results of this experiment suggest that AAI2-5 recognizes a cytoplasmic protein. We then separated MDA-MB-231 proteins using 10, 30, 50, and 100 kDa molecular weight cutoff filters. Proteins were quantified and 200 µg/mL of each sample was incubated with AAI2-5 for 30 min. Figure 6D shows that AAI2-5 was reactive with proteins with estimated molecular weight of above 50 kDa, but not inactive with the proteins with a moleculae weight below 50 kDa. This result suggests that the target of AAI2-5 has a molecular weight of more than 50 kDa. Fluorescence anisotropy analysis was also performed with the protein >50 kDa derived from 3 cell lines, MDA-MB-231, MCF10A and HEB. Figure S8 shows the corresponding titration curves. The data with MDA-MB-231 and MCF-10A can be fitted to the single binding site model, which shows that the apparent Kd of AAI2-5 for the protein from MDA-MB-231 is

Figure 6. Target of AAI2-5 is a protein. (A) Time course of fluorescence change of AAI2-5 cleavage reaction with untreated or protease digested MDA-MB-231 cell lysate. RF = relative fluorescence. (B) dPAGE analysis of AAI2-5 cleavage reaction with untreated or protease digested MDA-MB-231 cell lysate; NC = negative control; Unclv = uncleaved probe; Clv = cleaved probe. (C) dPAGE analysis of AAI2-5 cleavage reaction with whole protein, nuclear protein and cytoplasm protein of MDA-MB-231. (D) 10% dPAGE analysis of AAI2-5 cleavage reaction with different protein fractions of MDA-MB-231 cell lysate.

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Conclusion In this study, we have successfully applied the in vitro selection technique to derive RNA-cleaving fluorogenic DNAzyme (RFD) probes using the whole MDA-MB-231 cell lysate as the complex target. The RFD probes developed exhibit a high degree of sensitivity. Most of the RFD probes demonstrated substrate-cleavage abilities when incubated with MDA-MB-231 cell lysate, which could be used for characterizing the specific target in the cell line at the molecular level. The best probe among them, named AAI2-5, has excellent specificity as it is highly reactive with MDA-MB-231 but has much reduced activity towards normal cell lines, non-breast cancer tumor cell lines, and other subtypes of breast cancer cell lines. Moreover, AAI2-5 is able to identify breast tumors present in clinical specimens. Thus, this fluorogenic probe can be used to develop a simple “mix-and-read” fluorescence assay to achieve selective detection of breast tumors, which does not require expensive equipment and is easy to use. We have also found that AAI2-5 contains a highly conserved “TTAGCG” sequence motif that is essential for its activity. Preliminary experiments for target identification have revealed that the target that activates AAI2-5 is a protein (or proteins) localized to the cytoplasm of MDA-MB-231 cells with a molecular weight greater than 50 kDa. Our study shows RFD is a useful system for the generation of oligonucleotide probes for future use in breast cancer research and clinical diagnostics. This approach should allow better differentiation between molecular subgroups that exhibit distinct clinical behaviors and may enable the development of more effective individualized therapies.

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ASSOCIATED CONTENT

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Supporting Information Available

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This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION (20)

Corresponding Author *Corresponding author at: 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. Tel. /Fax. : + 86 755 26032094 E-mail address: [email protected]; [email protected]

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Author Contributions

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‡ These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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This work was supported by The Ministry of Science and Technology of China (2012ZX09506001-010), National Natural Science Foundation of China (No. 21302108 and No. 21402105) and Shenzhen Municipal government SZSITIC (No. KC2013ZDZJ0019A, JCYJ20130402145002379, and CXB201104210013A) and funding from the Canadian Institutes of Health Research (to YL).

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Analytical Chemistry

Table 1. Specificity test of representative probes to various breast cell linesa. MDA-MB-231

ZR-75-1

SK-BR-3

BT-474

MCF-7

BT-549

Hs 578Bst

MCF-10A

AAI1-1

++++

++

++

++

++

++

+++

++

AAI1-4

++++

++

++

++

+++

++

++

+++

AAI2-1

++++

+++

++

+

++

++

+++

+++

AAI2-4

++++

+++

+

+

+

+

++

+++

AAI2-5

++++

+

+

+

+

+

+

++

AAI2-6

++++

+++

++

+

+

++++

+++

+++

AAI2-9

++++

+++

++

+

+

+++

+++

+++

a The cleavage efficiency of each RFD candidate towards different breast cell lines was compared with MDA-MB-231. The percentage of probe cleavage was used to evaluate the specificity of these sequences: 85%: ++++. The final concentration of each sequence in reaction buffer is 50 nM.

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Table 2. Specificity test of representative probes to different cell linesa.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

breast

liver

cervical

glioblastoma

leukemia

colon

lung

normal

MDAMB-231

BEL7404

Hep G2

SKHEP-1

Hela

A172

U251

CCRFCEM

K562

HCT 116

NCIH69

HL-7702

AAI1-1

++++

+++

+++

+

++

+++

+++

++

+++

+++

+++

+++

AAI1-4

++++

+++

-

-

-

+++

+++

++

++

++

+

++

AAI2-1

++++

+++

++

++

++

++

+++

++

++

++

+

++

AAI2-4

++++

++

++

++

+

++

+

++

+

+

+

++

AAI2-5

++++

++

++

++

+

+

+

+

+

+

+

+++

AAI2-6

++++

+++

++

+

+

+++

++

++

+

++

+++

+++

AAI2-9

++++

+++

+++

++

++

++++

+++

+++

+++

++

++

++

a

The cleavage efficiency of each RFD candidate towards different cell lines was compared with MDA-MB-231. The percentage of probe cleavage was used to evaluate the specificity of these sequences: 85%: ++++. The final concentration of each sequence in reaction buffer is 50 nM.

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Analytical Chemistry

Table 3. Cleavage efficiency of AAI2-5 incubated with clinical specimens.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tumor Clv%

Malignant

Benign

N = 24

N = 13

2

1

15

10-35 %

9

6

2

35-60 %

8

5

0

60-85 %

2

1

0

> 85%

3

0

0

< 10% > 10%

Normal N = 17

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Tumor: 8.1% (3/37) Normal: 88.2% (15/17) Tumor: 91.9% (34/37)

Normal: 11.8% (2/17)

Analytical Chemistry

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For TOC only

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46x27mm (300 x 300 DPI)

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In vitro selection results and specificity of R28 DNA pool. (A) RNA-cleaving activity of different DNA pools in the presence of MDA-MB-231 cell lysate. Marker represents the expected cleavage product, which can be observed by fluorescence scan as it contains the fluorophore. Each DNA pool was incubated with 100 µg/mL MDA-MB-231 cell lysate for 30 min. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. (B) Activity of Round 28 pool towards different breast cell lysates. Hs 578Bst and MCF-10A are normal breast cell lines and others are tumor cell lines. (C) Activity of Round 28 pool towards lysates of nonbreast cell lines. HL-7702 is a normal cell line and others are tumor cell lines (see Materials and methods for details). 95x106mm (300 x 300 DPI)

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Characterization of AAI2-5. (A) P1/2 of AAI2-5 showed its catalytic activity with MDA-MB-231 cell lysate. The data was the average of three independent experiments. (B) AAI2-5 was incubated with 50 µg/mL of different breast cell lines lysate for 20 min. MCF-10A and Hs 578Bst are normal breast cells, while others are breast cancer cells. C, AAI2-5 was incubated with 50 µg/mL of different cell lines lysate for 20 min. Chang Liver, HL-7702 and QSG 7701 are normal human liver cell lines; HEB is normal human brain cell line; NCM460 is transverse colon cell line; 293T is human embryonic kidney cell line; others are cancer cell lines. NC was negative control, which was AAI2-5 incubated with 2× selection buffer plus lysis buffer for 20 min. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. 135x217mm (300 x 300 DPI)

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Sensitivity of AAI2-5 towards MDA-MB-231 cell lysate. (A) Real-time fluorescence monitoring over the background fluorescence. Protein concentration was considered for detection limits. (B) Rate of signal increase in response to the protein con-centration in MDA-MB-231 cell lysate. Inset shows the linear response at low protein concentrations. RF = Relative Fluorescence. The data is the average of three independent experiments. (C) 10% dPAGE and the corresponding 96-well microplate image of the probe incubated with MDA-MB-231 cell lysate (0-1000 µg/mL protein) for 60 min. Clv% = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. 101x122mm (300 x 300 DPI)

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Analytical Chemistry

Reactivity of AAI2-5 towards clinical specimens. (A) 10% dPAGE analysis of AAI2-5 incubated with clinical specimens. Each sample contained 50 nM probe and 100 µg/mL protein. Incubation time was 30 min for each group. Dark, grey and unfilled circles represent malignant breast tumor, benign breast tumor, paracarcinoma (or inflammatory tissue), respectively. No. = number of clinical specimens; Clv % = cleavage efficiency; Unclv = uncleaved probe; Clv = cleaved probe. (B) Statistics of data in Figure 4A. 71x29mm (300 x 300 DPI)

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Analytical Chemistry

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The sequence “TTAGCG” of AAI2-5 is important for its cleavage ability. (A) Mutant sequences of AAI2-5. The sequence motif “TTAGCG” of AAI2-5 was mutated into “TTTTTT” (AAI2-5-M), or deleted (AAI2-5-D). Each nucleotide of the motif was also mutated (AAI2-5-1, AAI2-5-2, AAI2-5-3, AAI2-5-4, AAI2-5-5 and AAI2-56). △ = the motif deleted; F, R and Q represent fluorescein-labeled dT, adenine ribonucleotide, and DABCYLlabeled dT, respectively. (B) Time-dependent fluorescence change of AAI2-5, the deletion sequence (AAI25-D) and mutated sequences (AAI2-5-1, AAI2-5-2, AAI2-5-3, AAI2-5-4, AAI2-5-5, AAI2-5-6, AAI2-5-M) incubated with 50 µg/mL of MDA-MB-231 cell lysate. The probe concentration was 25 nM. RF = Relative Fluorescence. (C) 10% dPAGE results of the sequences in panel A incubated with 50 µg/mL of MDA-MB-231 cell lysate for 6 h. NC = negative control; Unclv = uncleaved probe; Clv = cleaved probe. 83x97mm (300 x 300 DPI)

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Analytical Chemistry

Target of AAI2-5 is a protein. (A) Time course of fluorescence change of AAI2-5 cleavage reaction with untreated or protease digested MDA-MB-231 cell lysate. RF = relative fluorescence. (B) dPAGE analysis of AAI2-5 cleavage reaction with untreated or protease digested MDA-MB-231 cell lysate; NC = negative control; Unclv = uncleaved probe; Clv = cleaved probe. (C) dPAGE analysis of AAI2-5 cleavage reaction with whole protein, nuclear protein and cytoplasm protein of MDA-MB-231. (D) 10% dPAGE analysis of AAI2-5 cleavage reaction with different protein fractions of MDA-MB-231 cell lysate. 106x133mm (300 x 300 DPI)

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