Combinatorial Screening of DNA Aptamers for Molecular Imaging of

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Combinatory screening of DNA aptamers for molecular imaging of HER2 in cancer Guizhi Zhu, Huimin Zhang, Orit Jacobson, Zhantong Wang, Haojun Chen, Xiangyu Yang, Gang Niu, and Xiaoyuan Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00746 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Bioconjugate Chemistry

Combinatory screening of DNA aptamers for molecular imaging of HER2 in cancer

Guizhi Zhu†, Huimin Zhang†, ‡, Orit Jacobson†, Zhantong Wang†, Haojun Chen†, ┴, Xiangyu Yang†, ǁ, Gang Niu†, and Xiaoyuan Chen†,*

†

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland (MD), United States (USA)

‡

Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China

┴

Department of Nuclear Medicine, Xiamen Cancer Center, The First Affliated Hospital of Xiamen University, Xiamen, China, 361003 ǁ

Jiangsu Key Laboratory of Molecular Imaging and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009, China *

Corresponding Author: Dr. Xiaoyuan Chen Building 35A Rm GD937, 35A Convent Dr., Bethesda, MD 20892 Telephone: 301-451-4246 Email: [email protected]

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Abstract HER2, a cell membrane protein overexpressed in many types of cancers, is correlated with poor diagnosis, suboptimal treatment outcome, and low survival rate. Multiple HER2-targeted drugs have been developed for the treatment of HER2-overexpressing tumor, which can in turn downregulate HER2 expression. It is thus significant to profile HER2 expression for cancer prognosis, patient stratification, and monitoring therapy response. Aptamers, a class of single-strand DNA/RNA (ssDNA/ssRNA) ligands, are promising for molecular biomarker imaging. Aptamers typically have strong binding affinity, high selectivity, batch-to-batch reproducibility, and low toxicity, and systemically-injected aptamers often have high tumor-to-background ratio within a short time. However, current aptamers have been mostly screened in vitro, and these aptamers may lose binding ability in vivo due to conformational change under physiological environments. Here, a DNA library was combinatorially screened in vitro and in vivo, to select HER2-targeting DNA aptamers, termed as Heraptamers, and labeled with

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F for positron emission tomography

(PET) imaging of HER2 in ovarian cancer. Specifically, using Systematic Evolution of Ligands by EXponential enrichment (SELEX), Heraptamer candidates were first selected and validated in vitro using HER2 extracellular domain (ECD) and HER2-positive SKOV3 cancer cells; then, aptamer candidates were modified with alkyne, radiolabeled with

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F using azide-functionalized

precursors by click chemistry, and screened in SKOV3-tumor-bearing mice using PET. Two aptamers, Heraptamer1 and Heraptamer2, reached high tumor uptake ratios within as short as 1 h. At 1.5 h post injection, the tumor uptake ratio of these two aptamers was up to 0.5 %ID/g (injection dose/gram tissue), with tumor-to-muscle ratio of 4.55 ± 1.63 in SKOV3 tumor. In contrast, these aptamers have low uptake ratios in control MDA-MB-231 tumors. These preclinical studies showed that Heraptamers are promising for specific HER2 imaging. Key words: HER2, DNA aptamer, SELEX, radiolabeling, PET imaging


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Introduction Human epidermal growth factor receptor 2 (HER2, also known as Neu or ErbB2) is a member of the epidermal growth factor receptor (EGFR, also known as ErbB) family of receptor tyrosine kinases1. EGFRs mediate cell proliferation and differentiation during the development of embryos as well as adult tissues. HER2 is comprised of an ECD of about 630 amino acids, a transmembrane domain, and a cytoplasmic domain of tyrosine kinase. Ligand binding to an EGFR member other than HER2 induces receptor homo- or hetero- dimerization, during which HER2 is a preferred interaction partner. The dimerization further induces activation of the cytoplasmic kinase, leading to tyrosine autophosphorylation and initiation of downstream signaling pathways that contribute to cell proliferation and evasion from cell apoptosis2. HER2 is overexpressed in approximately 30% human ovarian cancer and breast cancer among other types of cancers, and the overexpression is correlated with poor prognosis, tumor metastasis, cancer relapse, resistance to chemotherapy, and poor survival rate3-4. HER2-target drugs, such as antibodies Trastuzumab and Pertuzumab, have been developed for cancer treatment5. Patient response to HER2-targeted therapeutics is correlated with HER2 overexpression, and HER2 expression level can be down-regulated by treatment with drugs5; whereas in patients without HER2 overexpression, these drugs are likely unbeneficial and even harmful. These clinical scenarios highlight the clinical significance of profiling HER2 expression level in cancer theranostics, such as cancer prognosis, patient stratification, and monitoring of therapy response. Molecular imaging is capable to quantify biomarker expression levels. Target-specific ligands range from small molecules, peptides and affibodies6, to macromolecules such as antibodies and aptamers. Aptamers are ssDNA/ssRNA that are screened via SELEX for specific binding of cognate targets, ranging from small molecules to macromolecules such as proteins7-14. Aptamers typically have ideal binding specificity and affinity15. The low molecular weight and 3 ACS Paragon Plus Environment


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negative charge of aptamers allow rapid penetration into target tissues and fast clearance from the blood, resulting in high signal-to-background ratio within a short time. Furthermore, DNA aptamers typically have high chemical stability, long shelf-lives, and can be manufactured with high batch-batch reproducibility. These characteristic features make aptamers attractive for molecular imaging16-22 of disease biomarkers, such as HER2. Even though a few aptamers for HER2 and other EGFRs have been reported23-27, a systematic preclinical HER2 imaging to evaluate binding specificity and sensitivity in animal models is highly desired. Many SELEX-based technologies have been developed to screen aptamers28. Protein-based SELEX can rapidly generate aptamers that specifically bind to a protein; yet purified proteins, especially membrane proteins like HER2, may not fully preserve natural conformations and cause loss of aptamers’ binding ability to natural proteins. To address this challenge, CellSELEX was developed to screen aptamers against membrane proteins at natural conformations on live cells29. Aptamers screened in vitro against disease biomarkers are able to bind to biopsied patient samples30. Yet, compared with target binding in vitro, in vivo aptamer binding may be affected due to pharmacokinetics and conformational variation under physiological environment, which may eventually disrupt the in vivo binding ability of aptamers selected in vitro. In vivo aptamer screening was reported9. However, aptamer screening thoroughly conducted in vivo is likely time-consuming and technically challenging, which hinders the wide application of this technology. For aptamer-based PET imaging, facile and efficient bioconjugation of aptamers with radiotracers is highly desired and has been pursued for a long time16, 19, 31, 32. Our group has recently developed multiple radiolabeling methods16, 19, among which a click-chemistry-based strategy involved only simple preparation and had outstanding synthesis yield using alkynemodified aptamers and azide-functionalized precursors.


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Here we present combinatory in vitro and in vivo screening of HER2-targeting ssDNA aptamers (termed as Heraptamers), and radiolabeling of Heraptamer candidates with

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F via click

chemistry for preclinical HER2 imaging in an ovarian tumor model. Specifically, in vitro proteinbased SELEX was first conducted using a HER2 ECD; next, the resulting DNA pool was subject to further screening by Cell-SELEX using HER2-overexpressing live SKOV3 cancer cells; finally, Heraptamer candidates was radiolabelled with

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F and further screened in vivo by PET imaging

in a SKOV3 tumor model. Aptamers selected from this combinatorial screening was validated using proteins and SKOV3 cells in vitro. The resulting aptamers were then studied for HER2 imaging in SKOV3 tumor, with HER2-negative MD-MBA-231 tumor as a control. Two aptamers, Heraptamer1 and Heraptamer2, enabled rapid visualization of HER2-positive tumors with high tumor-to-muscle ratio in preclinical PET imaging of HER2.

Results and Discussion In vitro aptamer screening To select DNA aptamers using SELEX, an ssDNA library was designed to have a 30-nucleotide random sequence flanked by two 18-nucleotide primer regions on 5' and 3'-ends, respectively (Figure 1C). This library will generate as many as 1.15 x 1018 (430) different DNA sequences to encode tremendous aptamer conformations. A pair of primers was optimized using NUPACK33 for efficient amplification of DNA products obtained from each round of SELEX. Biotin was modified on the 5' end of the antisense primer for ssDNA extraction from PCR products, and Alexa488 was modified on 5' end of the sense primer to monitor the binding activity of screened DNA pools. The PCR conditions were optimized to yield minimal by-products within the most PCR cycles to achieve both high yield and high purity of PCR products (Table S1). While protein-based SELEX is able to efficiently generate aptamers for target binding, Cell-SELEX can 5 ACS Paragon Plus Environment


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further ensure that aptamers bind to target molecules at natural conformation. Therefore, for in vitro aptamer screening, we combined protein-based SELEX, in which the DNA library was screened using HER2 ECD, and Cell-SELEX, in which the DNA pool generated from proteinbased SELEX was further screened using HER2-overexpressing live cells (Figure 1A). The ssDNA pool obtained from SELEX was then sequenced by 2nd-generation sequencing, and analyzed for sequence homology and frequency (Figure 1B). In protein-SELEX, His-tagged HER2 ECD [verified by Western Blotting (Figure S1)] was immobilized on Nickel-Nitrilotriacetic acid (NTA)-modified sepharose beads. HER2-ECDcoupled beads were washed and quantified immediately before screening. In the first round of SELEX, 100 pmole of HER2 ECD and 3.5 nmole DNA library were mixed and incubated in buffer at 37 °C for 1 h, followed by centrifugation and washing to remove unbound DNA. The resulting DNA-HER2-ECD complexes were subject to 3 steps of PCR: amplification PCR, optimization PCR, and preparative PCR. In amplification PCR, the above DNA-HER2-ECD complexes were PCR amplified for 10 cycles to enlarge the pool size; in optimization PCR, the products from amplification PCR were further amplified with a series of PCR cycles to optimize the PCR cycles to have minimal byproducts within the most PCR cycles (maximal yield and purity) by agarose gel analysis (Figure S2); in preparative PCR using the optimized cycle, the DNA products from amplification PCR was again amplified to prepare a DNA pool for the next round of screening. Cryo-concentrated dsDNA PCR products were incubated with streptavidinmodified agarose beads, and sense ssDNA was eluted using 1 M NaOH solution. The ssDNA products were desalted, lyophilized and quantified for the next round of screening. Proteinbased SELEX was repeated for 7 rounds, with 70 pmole HER2 equivalent and up to 200 pmole ssDNA as the starting pool. Blank sepharose beads and/or human serum albumin (HSA), an abundant protein in blood, were used for negative screening (Figure 1A) starting from Round 3,


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in order to remove DNA nonspecifically bound to blank beads or HSA. More than 200 pmole ssDNA products were generated from each round of protein-SELEX (Table S2). Following protein-SELEX, Cell-SELEX was performed using HER2-overexpressing live SKOV3 ovarian cancer cells (Figure S3). ssDNA pools were incubated with cells on ice for 1 h, and HSA was again added for negative screening. Unbound ssDNA or HSA-binding ssDNA were washed off, then DNA-cells complexes were scraped on ice, heat-inactivated, centrifuged, and the supernatant containing HER2-binding ssDNA was PCR amplified as above. Using flow cytometry, the binding ability of screened ssDNA pools to SKOV3 cells was found to be progressively increased (Figure 1D). The screening was stopped at the 7th round of Cell-SELEX when no further increase of DNA pool binding to SKOV3 cells was observed (Table S3). The final ssDNA pool was PCR ligated with DNA adaptors for sequencing (Table S4), and purified by gel extraction (Figure S4A). The purity of DNA pool was analyzed by electropherogram (Figure S4B, C). By 2nd-generation sequencing, about 2 million sequences were found in the resulting DNA pool (Figure S4D). These sequences were grouped based on homology, and aligned in the order of frequency. The most frequent 7 sequences (Table S5), denoted as Heraptamer1 to Heraptamer7, were chosen for in vitro characterization and in vivo screening.



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Figure 1. In vitro screening of Heraptamers for HER2 binding by SELEX. (A) Schematic illustration of in vitro screening for Heraptamers by SELEX. Negative selection using blank beads and HSA was utilized to ensure binding specificity of selected aptamers to HER2. Protein-based SELEX using HER2-ECDcoupled beads was conducted for 8 rounds, followed by 7 rounds of Cell-SELEX using live SKOV3 ovarian cancer cells. The DNA pool from the last round was subject to deep sequencing, followed by homology and frequency analysis to identify the most frequent sequences for downstream validation (B). (C) Sequences of library and primers used in SELEX. (D) Flow cytometry results showing the progressively increased binding of enriched DNA pools to target SKOV3 cells. DNA pools were labeled with Alexa488 via PCR using Alexa488-modified sense primer. SELEX was stopped at Round 15 when no further binding enhancement compared to the previous rounds was observed.


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In vitro characterization of aptamers Heraptamer1 to Heraptamer7 were first analyzed in silico. Even though one major motif with high homology was found within all these 7 sequences by sequence alignment (Figure S5), these 7 sequences had distinct secondary structures and dynamic conformations as revealed by structural simulation (Figure 2).

Figure 2. Simulated secondary structures of Heraptamer candidates. Structure simulation was performed using NUPACK (12). In each box, the left structure shows aptamer secondary structure with double-helix, the middle structure with nucleotide codes, and the right structure with equilibrium probability.

The above 7 aptamer candidates were then synthesized and tested in vitro for binding ability to HER2-ECD-coupled beads and SKOV3 cells. The binding ability was tested by flow cytometry using biotinylated aptamer candidates and streptavidin-PE-Cy5.5 conjugate. All 7 candidates showed enhanced fluorescence signal intensities upon incubation with HER2-coupled beads (Figure 3A), indicating their binding to HER2 ECD. Enhanced fluorescence intensity of live SKOV3 cells was also observed when cells were incubated with aptamer candidates on ice; in 9 ACS Paragon Plus Environment


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contrast, the original DNA pool did not bind to these cells, which validated the binding specificity to SKOV3 cells (Figure 3A). Since Cell-SELEX was performed on ice to prevent internalization of DNA into cells, and practical application of aptamers would be at physiological temperature, the binding ability of aptamers was further confirmed at 37 °C (Figure 3A). The specificity of these aptamer candidates were tested using HER2-negative cells, and none of these aptamer candidates bind to any of the tested HER2-negative MCF7, MDA-MB-435, and MDA-MB-231 cells (Figure 3B). Together, these results verified that these aptamers candidates only bind to HER2-ECD-coupled beads and HER2-overexpressing cells, but not to control beads or HER2negative cells, which demonstrate their specific binding to HER2. Using SKOV3 cells, the apparent dissociation constants (Kd) (13) of Heraptamers were determined to be at the low nanomolar range, which indicates their strong binding affinities (Table 1). Of note, these HER2targeted aptamers did not induce any significant cytotoxicity of SKOV3 cells after treatment for 2 days (Figure S6).

Figure 3. In vitro validation of Heraptamer candidates for selective HER2 binding. (A, B) Flow cytometry results suggest that Heraptamer candidates specifically bind to HER2–ECD-coupled beads, and target SKOV3 cells on ice and at 37 °C (A), but did not bind to nontarget HER2-negative MCF7, MDA-MB-435, or MDA-MB-231 cells (B). (DNA concentration: 200 nM). All DNAs were biotinylated, and streptavidin-PECy5.5 was used for fluorescence detection.


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Table 1. The apparent Kds of Heraptamer candidates. Data represent mean ± SD (n = 3). All DNAs were biotinylated, and streptavidin-PE-Cy5.5 was used for fluorescence detection.

Aptamer

Kd (nM)

Heraptamer1

5.1 ± 5.3

Heraptamer2

23.7 ± 11.2

Heraptamer3

8.9 ± 5.4

Heraptamer4

9.9 ± 5.6

Heraptamer5

7.4 ± 2.8

Heraptamer6

9.3 ± 3.2

Heraptamer7

7.9 ± 3.4

Radiolabeling of Heraptamer candidates After in vitro validation, the above aptamer candidates were then radiolabeled in order for in vivo screening in mice inoculated with HER2-positive tumor by PET imaging. Aptamer candidates were radiolabeled with positron emitted by

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F.

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F was chosen for PET because the half-life (109.8 min) of β+

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F is long enough for systemically administered aptamers, which are

typically cleared rapidly from circulation in vivo. Aptamer radiolabeling was conducted by click chemistry using alkyne-modified aptamers and an azide-functionalized precursor (Figure 4). This radiolabeling method was demonstrated to be able to fully convert alkyne-modified aptamers to

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F-radiolabelled aptamers16. The precursor,

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F-fluorobenzyl azide, was prepared

via spirocyclic iodonium ylide. This precursor recently proved very robust and reliable with high radiochemical yield and low de-fluorination. Specifically, first, a spirocyclic hypervalent iodine(III) precursor (1) was used for automated radiochemical synthesis of 11 ACS Paragon Plus Environment

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F-fluorobenzyl azide by


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aromatic fluoride substitution34; then, alkyne-modified aptamer candidates were radiolabeled with 18F using

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F-fluorobenzyl azide, via copper-mediated click chemistry.

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F-labeled aptamers

were purified using NAP-5 size-exclusion columns before use for PET imaging.

Figure 4. Radiolabeling of Heraptamers with

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F by click chemistry using alkyne-modified aptamers and

an azide-functionalized precursor. In the first step, a spirocyclic hypervalent iodine(III) precursor (1) was used to prepare radiolabeled with

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F-fluorobenzyl azide. In the second step, an alkyne-modified Heraptamer was

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F-fluorobenzyl azide using copper-catalyzed click chemistry in borate buffer 2+

supplemented with sodium ascorbate and Cu .

In vivo aptamer screening by PET imaging For in vivo aptamer screening,

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F-labeled aptamer candidates were intravenously (i.v.) injected

into SKOV3 xenograft tumor, followed by PET scans at 30 min post injection. A reported trivalent HER2-targeting aptamer, 3T2223, 35, was used for comparison. As shown in Figure 5A, the tumor uptake ratios of aptamer candidates in SKOV3 tumors varied, despite their comparable in vitro fluorescence binding signals on cells and HER2-ECD-coupled beads, likely due to the variation of aptamers’ conformational stability under physiological conditions. Aptamers were rapidly cleared mainly through the renal route, resulting in high radioactivity in 12 ACS Paragon Plus Environment

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bladders and kidneys. Gallbladder, which is involved in the metabolism of nucleic acids, also showed strong radioactivity presumably as a result of aptamer metabolism in the body. The tumor uptake ratios of aptamers were quantified based on PET images (Figure 5B). Among all 8 tested ligands, Heraptamer1 and Heraptamer2 had relatively high tumor uptake. Thus, Heraptamer1 and Heraptamer2 were selected for further study.

Figure 5. In vivo screening of Heraptamer candidates. (A) Representative coronal (upper) and transverse (lower) PET images displaying SKOV3-inoculated mice i.v. injected with

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F-labeled Heraptamer

candidates and 3T22, a reported HER2-targeting aptamer as comparison, at 30 min post injection. White arrow heads mark the tumors, and red arrow heads mark the gallbladder if discernable. (B) Quantitative comparison of the tumor uptake ratios of Heraptamer candidates and 3T22. Data represent mean ± SD; n= 3. *P ≤ 0.05, **P ≤ 0.01 (Student’s t-test). ID/g: injection dose/gram of tumor tissue.

Using Heraptamers for PET imaging of HER2 in an ovarian cancer mouse model 18

F-labeled Heraptamer1 and Heraptamer2 were further studied for PET imaging of HER2 in

both HER2-positive SKOV3 tumor and HER2-negative MDA-MB-231 tumor (HER2 expression verified in Figure S3). Further, the in vitro HER2-specific binding ability of Heraptamers was verified by an “aptamer blotting” assay using HER2 ECD (Figure S7A), as well as by confocal 13 ACS Paragon Plus Environment


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microscopy using SKOV3 cancer cells (Figure S7B). For PET imaging,

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F-radiolabeled

aptamers were again i.v. injected into tumor-bearing mice, followed by PET scan at 15 min and 1 h post injection (Figure 6A). In SKOV3 tumor, at 15 min post injection, high tumor uptake was observed for both Heraptamer1 (1.04 ± 0.18%ID/g) and Heraptamer2 (0.67 ± 0.10%ID/g); at 1 h post injection, the tumor uptake was decreased to 0.52 ± 0.04%ID/g for Heraptamer1 and 0.41 ± 0.14 %ID/g for Heraptamer2. Blocking

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F-radiolabeled aptamers with the corresponding

unlabeled aptamers dramatically reduced the tumor uptake ratios in SKOV3 tumors (Figure 6A), suggesting in vivo binding specificity of these aptamers to HER2. At 1.5 h post injection, tumor and major organs were collected, and the biodistribution of aptamers in these organs was determined by ɣ-counting (Figure 6B). The tumor-to-muscle ratios were 3.37 ± 1.04 for Heraptamer1, and 4.55 ± 1.63 for Heraptamer2 (Figure 6C). The ability of these aptamers to exhibit high signal-to-background (tumor-to-muscle) ratios within a short time may help to facilitate clinical diagnosis, patient stratification, or monitoring therapy response. It is also noteworthy that, the ability of imaging within a short time would avoid the otherwise complication caused by nucleic acid degradation, because ssDNA aptamers were reasonably stable within this time period as demonstrated previously16, 19. In mice inoculated with HER2-negative MDAMB-231 tumor, PET imaging at 60 min post injection of

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F-radiolabeled aptamers determined

the tumor uptake ratios to be 0.15 ± 0.04 %ID/g and 0.18 ± 0.05 %ID/g for Heraptamer1 and Heraptamer2, respectively (Figure 6D-E). Thus, the specificity of these two aptamers for in vivo HER2 imaging was evidenced by both the low tumor uptake ratios in HER2-negative MDA-MB231 tumor and the reduced tumor uptake ratios by blocking using the corresponding unlabeled aptamers. Taken together, these preclinical studies provide evidence of using Heraptamer1 and Heraptamer2 for HER2 imaging.


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Figure 6. Using Heraptamer1 and Heraptamer2 for specific HER2 imaging in tumor-inoculated mouse models. (A) Representative coronal (upper) and transverse (lower) PET images displaying SKOV3 tumorinoculated mice i.v. injected with (middle) post injection, and

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F-labeled Heraptamer1 and Heraptamer2 at 15 min (left) and 60 min

F-labeled aptamers with the corresponding unlabeled aptamers for blocking

at 60 min post injection (right). White arrow heads mark the tumors, and red arrow heads mark the gallbladder if discernable. (B) Biodistribution of Heraptamer1 and Heraptamer2 in SKOV3-inoculated mice at 1.5 h post injection. (C) Tumor-to-muscle ratios of SKOV3-inoculated mice injected with

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F-labeled

Heraptamer1 and Heraptamer2 at 1.5 h post injection. (D) Representative coronal and transverse PET



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images displaying MDA-MB-231 tumor-inoculated mice injected with

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F-labeled Heraptamer1 and

Heraptamer2 at 60 min post injection. (E) Tumor uptake ratio of Heraptamer1 and Heraptamer2 in HER2negative MDA-MB-231 tumor at 1 h post injection. White arrow heads mark the tumors in (A) and (D). Data in (B, C) represent mean ± SD (n = 5).

Conclusion By combinatory in vitro protein-based SELEX using HER2 ECD, in vitro Cell-SELEX using live HER2-overexpressing cells, and in vivo screening using radiolabeled aptamer candidates by PET imaging in HER2-overexpressing tumor models, we selected Heraptamers as HER2binding ligands. Heraptamer candidates were radiolabelled with

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F via an efficient click-

chemistry-based method using alkyne-modified DNA and an azide-functionalized precursor. As a result of combinatory screening, Heraptamer1 and Heraptamer2 were selected given their relatively high tumor uptake ratios. Preclinical studies demonstrate that Heraptamer1 and Heraptamer2 were capable of rapid and specific HER2 imaging in a SKOV3 ovarian cancer model, with HER2-negative MDA-MB-231 tumor model as a control. These results suggest that Heraptamer1 and Heraptamer2 are promising ligands for HER2 imaging in cancer.

Materials and Methods Protein-based SELEX His-tagged HER2 ECD (SKU: YCP1045) was purchased from SPEED BioSystems (Gaithersburg, MD). Reconstituted protein in PBS was verified by western blotting. Sepharose beads (GE Healthcare Life Sciences, Pittsburgh, PA) were washed using PBS supplemented with 5 mM imidazole for 3 times before use. HER2 ECD was coupled onto washed beads by 16 ACS Paragon Plus Environment

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incubating HER2 ECD and beads in PBS supplemented with 5 mM imidazole, and the mixture was vortexed for 1 h at room temperature. The resulting mixture was washed using PBS supplemented with 5 mM imidazole for 3 times to remove free proteins. Removed free proteins were quantified by absorbance at 280 nm, based on which the coupling efficiency of HER2 ECD on beads was calculated. HER2-ECD-coupled beads and DNA library were used for protein-SELEX to screen HER2targeting DNA aptamers. DNA library was first snap-cooled by denaturing at 95 °C for 5 min, then immediately cooled down on ice for 5 min, followed by recovery for 1 h at room temperature, in order to allow the formation of the optimal conformation. For each round of positive screening, snap-cooled DNA pools and HER2-ECD-coupled beads (see amount in Table S2) were mixed, incubated, and vortexed in PBS supplemented with 5 mM Mg2+ and 5 mM imidazole for 1 h at room temperature. HER2-DNA complex on beads were collected by centrifugation, followed by washing for 3 times. The resulting complexes were then directly subject to 3 steps of PCR amplification under optimized condition (Table S1): 1st PCR for amplification of DNA pools, 2nd PCR for further optimization of PCR cycles, and 3rd PCR for preparation of maximal DNA products using the optimized PCR condition from the 2nd step. The PCR products were then mixed with streptavidin-agarose beads, filtered and washed off free DNA or proteins, and finally the sense strand ssDNA was eluted using NaOH solution (1 M). ssDNA solution was desalted, lyophilized, and quantified for the next round of SELEX. For negative screening, blank Sepharose beads were incubated with DNA pools under the same condition for 20 min, followed by centrifugation and collection of the supernatant. The supernatant was then used for positive screening. Additionally, negative selection using HSA was conducted by adding HSA (1 mg/mL) directly into the DNA-HER2 mixture during positive screening.



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Radiolabeling Heraptamer candidates were radiolabeled with was employed for

18

F for PET imaging. Click chemistry conjugation

18

F radiolabeling as reported before (11). Briefly, a spirocyclic hypervalent

iodine(III) precursor (1) was used for automated radiochemical synthesis of

18

F-fluorobenzyl

azide by aromatic fluoride substitution in DMF (120 ºC, 10 min), on an automated modular system (Eckert & Ziegler Eurotope GmbH). Aptamer candidates were labeled with an alkyne group on the 5'-end, and they were then radiolabelled with

18

F using an

18

F-fluorobenzyl azide,

via copper-mediated click conjugation in borate buffer supplemented with Cu2+ and sodium ascorbate (pH 8.6). The radiolabeling was verified by LC-MS analysis. The radiolabeled aptamers were purified from crude reaction mixture using NAP5 columns, and fraction 4 (250 µL per fraction) was collected as the product, which was directly used for PET imaging. PET imaging Mice bearing SKOV3 tumor or MDA-MB-231 tumor were anesthetized using isoflurane/O2 (2% v/v) before injection. Anesthetized mice were injected through tail vein with

18

F-labeled

Heraptamers (4.44-5.55 MBq/120-150 µCi per mouse) in PBS (100 µL). At specified time points post injection, mice were scanned on an Inveon DPET scanner (Siemens Medical Solutions, Malvern, PA). PET images were reconstructed without correction for attenuation or scattering using a three-dimensional ordered subsets expectation maximization algorithm. ASI Pro VMTM software was used for image analysis. Regions of interest (ROI) were drawn on tumors to calculate the %ID/g. Biodistribution of Heraptamers


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The above mice used for PET imaging were euthanized at 1.5 h post injection. Organs and blood were collected and wet-weighed. The collected organs and blood, together with a series of standard solution, were measured for

18

F radioactivity on a gamma-counter (Wallac Wizard

1480, PerkinElmer). The radioactivity of organs and blood was converted to be the percentages of the injected dose per gram of tissue (%ID/g).

Note The authors declare no competing financial interest.

Acknowledgment This work was supported by intramural research program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH).

Supporting Information Supporting Information includes 1) supplemental methods and materials, 2) additional figures showing western blotting, schematic SELEX work flow and agarose gel electrophoresis, HER2 overexpression on SKOV3 cells, Ion-Torrent 2nd generation sequencing and analysis, determination of Kd, cell viability of SKOV3 cells treated with Heraptamers, and in vitro Heraptamer2 binding to HER2, and 3) additional tables showing PCR conditions, summary of HER2 protein-SELEX and Cell-SELEX, DNA sequences used for 2nd-gen high throughput sequencing, and sequences of the most frequent Heraptamer candidates. Supporting Information is available free of charge on the ACS Publications Website.

Abbreviation ECD: extracellular domain EGFR: epidermal growth factor receptor HER2: Human epidermal growth factor receptor 2 19 ACS Paragon Plus Environment


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HSA: human serum albumin NTA: Nickel-Nitrilotriacetic acid PET: positron emission tomography ROI: region of interest SELEX: Systematic Evolution of Ligands by EXponential enrichment %ID: percent of injected dose %ID/g: percent of injected dose per gram of tissue


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TOC


Combinatorial Screening of DNA Aptamers for Molecular Imaging of

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