Aptamer-based cell imaging reagents capable of fluorescence ......This journal is' The Royal ociety of Chemistry 2014 Chem. Commun., 01 50, 1--1 | 12329 Cite this Chem. Commun., 2014,

This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 12329--12332 | 12329

Cite this:Chem. Commun., 2014,

50, 12329

Aptamer-based cell imaging reagents capable offluorescence switching†

Yun Kyung Jung,ab Min-Ah Woo,a H. Tom Soh*c and Hyun Gyu Park*a

We describe an aptamer-conjugated polydiacetylene imaging probe

(ACP) that shows highly specific fluorescence switching upon binding

to epithelial cancer cells that overexpress the tumor biomarker protein

EpCAM (epithelial cell adhesion molecule) on their surface.

Fluorescence imaging of tissue and cell surface markers is awidely used technique in many areas of biology and medicine.1–5

Typically, such imaging is achieved with fluorophores conjugatedto an affinity reagent (e.g. a monoclonal antibody, a peptide or anaptamer) that specifically binds to the surface marker of interest.2

In order to achieve high-contrast imaging, it is imperative thatthe affinity reagent binds specifically to the surface marker withhigh affinity, and that the fluorophore is bright and photostable.Ideally, the fluorophore should exhibit low background fluores-cence when not specifically bound to the surface marker, such thata visible signal is only generated in the bound state. Unfortunately,this is a major challenge for most fluorophores, for which thefluorescence intensity and wavelength remain constant regard-less of their binding state.

Active probes capable of changing their fluorescence state –for example, switching from a ‘‘dark state’’ when unbound to a‘‘bright state’’ when specifically bound to their target – offer ameans for obtaining dramatically improved imaging contrast.Recent years have witnessed growing interest in ‘‘switchable’’fluorescence reporters such as Dronpa,3 cyanine dyes,4 andpolydiacetylenes (PDA).5 In particular, PDAs – a family of

nanoscale, conjugated polymers synthesized by polymerizingmonomeric diacetylene lipids through UV irradiation – offermany advantages, because they are photostable, exhibit lowcytotoxicity, and can be readily conjugated to a wide range ofbiopolymers.5 A number of recent publications have reportedPDAs capable of switching their fluorescence state in responseto various external stimuli. For example, Kim et al. developedmicropatterned PDAs that undergo fluorogenic transitions uponthermal stress or interaction with cyclodextrin.5a,b Our group showedthat the fluorescence of streptavidin-functionalized PDAs canbe modulated upon binding to biotinylated DNAs.5c Importantly,Jelinek et al. have reported that the fluorescence of PDA attachedonto live cell surfaces changes when it non-specifically interacts withcell membrane-perturbing molecules.5d–f Although these pioneeringexamples have blazed a promising trail, maximizing the utilityof PDA probes will require a novel class of probes that do notrespond when they non-specifically interact with cell surfaces,and only switch their fluorescence state when they bind tospecific cell surface makers. To the best of our knowledge, suchPDA probes have not yet been demonstrated.

Toward this end, here we describe an active PDA imagingprobe that switches its fluorescence state when it binds to aspecific cell-surface marker. Our probe is functionalized with ahigh-affinity DNA aptamer that has been selected for specificbinding to a cell-surface marker of interest. Our aptamer-conjugated PDA (ACP) probe is prepared by incorporatingaptamer-conjugated diacetylene monomers into a diacetyleniclipid matrix comprising dimyristoyl phosphatidylethanolamine(DMPE) and 10,12-pentacosadiynoic acid (PCDA).6 In the unboundstate, our ACP stays ‘‘dark’’ in the red-channel because the conjugated(ene–yne) polymer backbone remains unchanged (no emissionpeak) (Scheme 1, top). In contrast, when the ACP probe binds toits target surface marker, it switches to a ‘‘bright’’ state in the red-channel (emission peak at 563 nm) because the conjugated back-bone of PDA undergoes a conformational transition that causes ared-shift in its fluorescence (Scheme 1, bottom). As a model target,we chose the epithelial cell adhesion molecule (EpCAM) protein;EpCAM is a tumor-specific antigen for malignancies of epithelial

a Department of Chemical and Biomolecular Engineering (BK21 + Program), KAIST,

291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.

E-mail: [email protected] School of Natural Science, UNIST, UNIST-gil 50, Eonyang-eup, Ulju-gun,

Ulsan 689-798, Republic of Koreac Materials Department, Department of Mechanical Engineering, University of

California, Santa Barbara, CA 93106, USA. E-mail: [email protected]

† Electronic supplementary information (ESI) available: Experimental details, thescheme of aptamer selection and conjugation with a diacetylene monomer,EpCAM binding analysis after one round of selection, Kd values of representativeaptamer sequences for EpCAM, epithelial cancer cell imaging by a JYK-02 ACPprobe, and SEM and DLS analysis of the JYK-01 ACP probes after protein addition.See DOI: 10.1039/c4cc03888f

Received 21st May 2014,Accepted 21st August 2014

DOI: 10.1039/c4cc03888f

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lineage, and is used as a marker for circulating tumor cells(CTCs).7 We show that our ACP probes enable specific visuali-zation of epithelial cancer cells by switching their fluorescenceemission from nonfluorescence (blue state) to fluorescence(lem = 563 nm, red state) upon binding the EpCAM moleculespresent on the cell surface.

Although RNA and DNA aptamers for EpCAM have beenpreviously reported,8 we selected our own DNA aptamers toachieve higher specificity by utilizing the microfluidic SELEX(M-SELEX) method previously described by our group(Scheme S1†).9 The efficiency of our SELEX process was verifiedafter one round of positive selection (Fig. S1†). We performedthree rounds of positive selection using EpCAM-coated magneticbeads and one round of negative selection using bovine serumalbumin (BSA)-coated magnetic beads to obtain an enriched aptamerpool (see the ESI†). We then measured the binding affinity ofthe enriched pool for EpCAM via a fluorescence-based bead-binding assay.9 Assuming Langmuirian binding betweenEpCAM and the aptamers, we measured the average equili-brium dissociation constant (Kd) of the enriched pool to be8.4 � 2.2 nM (Fig. 1A, ). These aptamers showed negligiblebinding to BSA-coated or uncoated carboxyl magnetic beads(Fig. 1A, m and ).

We obtained sequences for individual EpCAM aptamers bycloning the enriched aptamer pool into the TATA cloning vectorand transforming into E. coli (see the ESI†). From the fiftyrandomly picked clones, we identified three clusters thatshowed notable sequence similarity. We selected representativesequences from each cluster and measured their Kd (see theESI† for experimental details). Aptamers from all three clusters

showed Kd in the low nanomolar range (Fig. S2†); the aptamerwith the highest affinity (JYK-01; 50-TGAAGGTTCGTTGTTTCGGTGGGTGTAGACTCTTTAGAAGAGATACAGATTTTGGGAATG-30)exhibited a Kd of 8.6 � 2.5 nM. The binding affinity of thisaptamer was higher than previously described EpCAM aptamers8

and comparable to engineered anti-EpCAM antibodies10 (Fig. 1B).We synthesized ACPs by modifying the 50 ends of our JYK-01

aptamers with an amine group and conjugating them to diacetylenemonomers (10,12-tricosadiynoic acid; TCDA) through EDC–NHScoupling chemistry (Scheme S1, ESI†). In order to verify the func-tionality and specificity of our ACP probe, we measured the fluores-cence after incubating the ACP with EpCAM in solution (Fig. 1C;excitation at 485 nm, emission at 563 nm).5e,f We simultaneouslytested the specificity of our ACP probes by challenging them withtwo unrelated proteins, bovine serum albumin (BSA) and immuno-globulin G (IgG). Compared to the strong fluorescence signal fromEpCAM, BSA and IgG produced negligible signals. To furtherconfirm that the fluorescence change is indeed the result of specificbinding between EpCAM and the JYK-01 aptamer, we fabricated anACP containing a random sequence (50-TAAGTATATCGTGCCTGCGACTATGTTATGATGAGGCAGT CTTTAACCTGACTCGTAATA-30).The resulting ACP probes exhibited a negligible fluorescence signalwhen challenged with EpCAM (Fig. 1C), adding further evidence thatthe change in fluorescence requires specific binding between JYK-01and EpCAM. To quantify the sensitivity of our ACP probe, wegenerated a calibration curve by plotting the change in fluorescenceas a function of EpCAM concentration (Fig. 1D). We characterizedthe limit of detection (LOD) using the 3sb/m criterion,11 wheresb is the standard deviation of a negative control (blank) and mis the slope in the linear range. Using this metric, we estimateda LOD of B1 ng mL�1 for our sensor.

Scheme 1 Epithelial cancer cell imaging utilizing fluorescent polydi-acetylene (PDA) molecular probes conjugated to an EpCAM aptamer.

Fig. 1 (A) We used a fluorescence-based bead assay to measure thebinding affinity of the enriched aptamer pool. After three rounds of positiveselection with EpCAM and a single round of negative selection with BSA,aptamers showed nanomolar affinity for EpCAM with negligible binding toBSA-coated or uncoated beads. (B) We used the same assay to determinethe binding affinity of the JYK-01 aptamer. (C) Specificity analysis of JYK-01- orrandom sequence-functionalized ACPs for EpCAM protein. Each ACP wasincubated for 1 hour with a protein-free control (Con) or 4 ng mL�1 of EpCAMor non-target proteins BSA and IgG. (D) Quantitative fluorescence detection ofJYK-01-conjugated ACPs as a function of EpCAM concentration.

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We tested the capacity of the ACP probes to switch fluorescenceupon binding to EpCAM on cell surfaces (Fig. 2). As target cells, weused the human colon adenocarcinoma cell line HT-29, which hasan epithelial morphology and is known to express EpCAM.12 Weverified EpCAM expression by incubating these cells with an AlexaFluor 555-labeled anti-EpCAM antibody, and observed strongfluorescence (Row 1, left). We used two different cells as negativecontrols: normal human fibroblast cell line WI-38, derived froma non-tumorigenic epithelial lineage, and melanoma cell lineWM-266-4, which originated from a different non-epithelial lineage.Both cell types showed negligible fluorescence when incubatedwith the same antibody (Row 1, center and right). In addition, wemeasured the fluorescence from ACPs conjugated to random DNAsequences as negative controls and did not observe any appreciablefluorescence signal for any of the three cell lines (Row 2). Critically,ACP probes conjugated to the JYK-01 aptamer specifically labeledHT-29 cells expressing EpCAM with strong fluorescence intensitycomparable to that seen with the antibodies (Row 3, left). The sameACP probes exhibited negligible fluorescence when incubated witheither of the EpCAM-negative cell lines, WI-38 (Row 3, center) orWM-266-4 (Row 3, right).

Several recent studies have investigated the underlyingmechanism behind the observed fluorescence switching,5b,g–j,13

and much of the evidence suggests that this switching is theresult of mechanical stress on the PDA structure caused bybinding to EpCAM. Such external stresses on the PDA structureare known to trigger electronic transitions in the delocalizedp-electron networks of the PDA backbone and thereby changethe optical properties. In this model, radiative transition (decay)from a higher excited Ag symmetry state (dipole-forbidden) to

the lowest excited Bu state (dipole-allowed) is believed to beresponsible for shifting the fluorescence wavelength from short(blue) to long (red).5b,g–j,13 We would therefore suppose that directmechanical interaction between the ACP probes and EpCAM is anecessary requisite for fluorescence switching to occur.

To further support this model for fluorogenic transitions ofACP probes, we investigated changes in probe morphology andsize before and after the addition of EpCAM via SEM and DLS.6

We observed that in the absence of EpCAM, ACP probes aremonodisperse and spherical in shape with an approximatediameter of B364 nm (Fig. S4a and d†). However, upon additionof EpCAM, the ACP probes aggregate and show a more denseinterior structure, with sizes ranging up to B933 nm (Fig. S4band d†). Adding an unrelated protein (i.e., IgG) does not triggerthe same changes in size and morphology (Fig. S4c†). Theseresults add further evidence that fluorescence switching isindeed caused by physical stress and morphological changesin the PDAs caused by binding to EpCAM on the cell surface.

In summary, we report an active aptamer-conjugated PDAprobe that switches its fluorescence when specifically bound toa targeted cell-surface marker. We discovered DNA aptamersthat bind to the EpCAM protein and used these to synthesizeACP probes that enable specific visualization of EpCAM-positivecells by switching their fluorescence emission wavelength. Giventhat aptamers are available for a broad range of surface markers,and PDA nanoparticles offer facile synthesis, low cytotoxicity,5d,f

and excellent photostability,14 we believe that a deeper under-standing of fluorescence-switching mechanism could lead tonext generation of smart imaging probes with higher specificity,signal intensity and contrast.

Fig. 2 Epithelial cancer cell imaging, using ACP probes that target overexpressed EpCAM protein at the cell surface. Bright-field (left) and confocalfluorescence (right) microscopy images of HT-29 (epithelial cancer), WI-38 (normal epithelial; EpCAM-negative), and WM-266-4 (human melanoma;EpCAM-negative) cells. The images were taken after 2 hour incubation with (Row 1) Alexa Fluor 555-labeled anti-EpCAM antibody, (Row 2) ACP with arandom aptamer, and (Row 3) ACP with JYK-01. Nuclei were labelled with DAPI (blue). Scale bar = 20 mm.

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This research was supported by Basic Science ResearchProgram through the National Research Foundation ofKorea (NRF) funded by the Ministry of Education, Scienceand Technology (NRF-2012R1A1A3015259). We are also gratefulfor the financial support of Center for BioNano Health-Guardfunded by MSIP of Korea as Global Frontier Project (GrantH-GUARD_2013M3A6B2078964). HTS is grateful for the supportfrom ARO Institute for Collaborative Biotechnologies (W911F-09-D-0001), National Institutes of Health (U54 DK093467,U01HL099773-01, R01A1076899), and Department of Defense(W81XWH-09-0698).

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