Smart Dual Quenching Strategy Enhances the Detection Sensitivity of

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A Smart Dual Quenching Strategy Enhances the Detection Sensitivity of Intracellular Furin Zijuan Hai, Jingjing Wu, Dilizhatai Saimi, Yanhan Ni, Rongbin Zhou, and Gaolin Liang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05251 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Analytical Chemistry

A Smart Dual Quenching Strategy Enhances the Detection Sensitivity of Intracellular Furin Zijuan Hai,† Jingjing Wu,† Dilizhatai Saimi,† Yanhan Ni,† Rongbin Zhou,‡ and Gaolin Liang†,* † CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡ Institute of Immunology and the CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS center for Excellence in Molecular Cell Sciences, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei 230027, China

ABSTRACT: Development of sensitive fluorescence “Turn-On” strategies for imaging enzyme activity in living cells is of disease-diagnostic importance but remains challenging. Herein, by employing a click condensation reaction and rational design of a single quenched probe Cys(StBu)-Lys(Gly-Lys(DABCYL)-Gly-Gly-Arg-Arg-Val-Arg-Gly-FITC)-CBT (1), we developed a “smart” dual quenching strategy and applied it to detect intracellular furin activity with enhanced sensitivity. At physiological condition, 1 was subjected to reduction-controlled condensation reaction to form 1-NPs and its fluorescence intensity further dropped to 1/2.8 of its original. Upon furin cleavage in vitro, the dual quenched 1-NPs had fluorescence “Turn-On” contrast 11 folds more than that of single quenched control probe FITC-Gly-Arg-Val-Arg-Arg-Gly-Gly-Lys(DABCYL)-Gly-OH (1-P). Live cell imaging results indicated that 1 showed fluorescence “Turn-On” contrast 6.3-fold of that of 1-P for sensing intracellular furin activity. We envision that, by replacing the RRVR substrate with other enzyme-cleavable ones, our versatile “smart” dual quenching strategy could be easily adjusted for the detection (or imaging) of other intracellular enzymes’ activity with enhanced sensitivity.

Fluorescence is always favorably applied for the detection of important analytes or noninvasive visualization of biological processes in real time due to its high sensitivity, technical simplicity, cost-effectiveness, and fast processing time.1 From the aspect of signal property, fluorescence probes are roughly classified into three types: “Always On”, “TurnOn”, and “Turn-Off”. Among them, fluorescence “Turn-On” probes are advantageous over the other two types due to their lowest background noise.2-6 Before the probes “Turn-On” at the targeting site, their signal needs to be turned “Off” and two prevalent strategies have been widely used for this purpose (i.e., fluorescence resonance energy transfer (FRET) based on dye-dark quencher systems and aggregation-caused quenching (ACQ) based on dye-dye systems).7 Here, we denote the “Turn-On” probes that use one of above two strategies as single quenched probes and those “Turn-On” probes using above both strategies as dual quenched probes. Obviously, compared with single quenched probes, dual quenched ones can further reduce the background signal intensity and therefore the magnification times of their “Turn-On” fluorescence signal are much larger. To date, several dual quenched nanoprobes have been designed for the imaging of protease activity with high sensitivity.8-11 In these studies, fluorescent dyes were linked to dark quenchers with protease-cleavable peptides and then the dye-peptide-quencher complexes were covalently conjugated to the premade nanoparticles. Conjugation of the complexes to the nanoparticles resulted in another dye-dye quenching effect and therefore the dyes on the nanoparticles are dual quenched. Nevertheless, besides the difficulty and reproducibility of their

fabrications, these premade dual quenching nanoprobes were also facing the problems of cell membrane translocation and targeting when applied for cell imaging.12 Compared with nanoprobes, cellular uptake of small molecular probes is faster and easier. But to the best of our knowledge, using a single quenched small molecular probe to “smartly” self-assemble into dual quenched nanoprobe inside cells has not been reported. In 2010, Rao and co-workers developed a biocompatible, click condensation reaction between the 1,2-aminothiol group of cysteine (Cys) and the cyano group of 2cyanobenzothiazole (CBT) which could be applied to selfassemble nanostructures in living cells under the control of pH, reduction, or protease.13-15 Up to now, this reaction has shown promising applications in protein labeling,16-17 molecular imaging (optical imaging,18 magnetic resonance imaging,19-20 and positron emission tomography imaging21-22), intracellular hydrogelation,23 cell bridging,24 drug release,25 etc. Inspired by above studies, as shown in Figure 1A, in this work, we rationally designed a single quenched small molecular fluorescence probe 1 which instantly and “smartly” self-assembled into dual quenched nanoparticles 1-NPs inside cells under reduction-controlled condensation. Under the proteolytic hydrolysis of intracellular furin which preferentially cleaves Arg–X–Lys/Arg–Arg ↓ X peptide substrates,26-29 the dual quenched fluorophore FITC was detached from the nanoparticle which resulted in fluorescence “Turn-On”. Because the trans-Golgi protease furin is a tumoroverexpressing protein convertase, sensitive detection of furin with 1 offers a practicable strategy for the early diagnosis of

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certain cancerrs. This “smartt” compound C Cys(StBu)-Lyss(GlyLys(DABCYL L)-Gly-Gly-Argg-Arg-Val-Arg--Gly-FITC)-CB BT (1) for intracellulaar dual quenchhing was desiggned containinng the following com mponents: (1) a CBT motif annd a disulfidedd Cys motif for reduuction-controlleed CBT–Cys condensation; (2) a DABCYL struucture as a daark quencher of the fluoropphore FITC; (3) an Arg-Arg-Val-A Arg tetrapeptide sequence w which a is not only improoves the cellullar uptake of thhe probe but also the substrate for furin cleavvage; (4) an eextending dipeeptide Gly-Gly to aboove tetrapeptidde for avoiding the steric hinddrance to furin cleaavage. Its inttracellular duual quenching and subsequent ffurin-instructedd florescencee “Turn-On” are schematically illustrated in F Figure 1A. In ddetail, after enttering cancer cells, single quenchhed 1 will suubject to reducctioncontrolled conndensation and self-assemble into dual quennched nanoparticles (i.e., 1-NPs) with additioonally loweredd the fluorescence intensity. Unnder the speecific cleavagge of C-GRVRR will be detached from intracellular fuurin, free FITC the nanoparticcle and its duall quenched fluuorescence is totally relieved, thus achieving fluoorescence “Turrn-On” with a high signal-to-noisee (S/N) ratio. To validate our hypothesiis, as shown in Figuure 1B, the sinngle quenched precursor of 1 (i.e., FITC-Gly-Argg-Val-Arg-Arg--Gly-Gly-Lys(D DABCYL)-Glyy-OH, 1-P), which could not self-assemble innto dual quennched nanoparticles but is also suusceptible to ffurin cleavage, was used as a posittive control proobe.

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nannoparticles (1-N NPs) (A) or ssingle quenchedd small molecule conntrol probe 1-P (B) for the senssing of furin acttivity.

W We began the study with thhe syntheses oof precursor 1-P, proobe 1, and FITC-GRVRR (Schemes S1-S3). These thrree com mpounds were fully characterized using 1H NMR, 13C NM MR, maass (MS), and UV-Vis specttral analyses (F Figures S1-S12). Aft fter syntheses, we w firstly usedd tris(2-carboxyyethyl) phosphiine (TC CEP) to triggeer the condensation of 1 to self-assemble s tthe duaal quenched naanoparticles (1-NPs) in vitro.. In detail, 25 μ μM 1 iin furin buffer (100 mM HEP PES, 1 mM CaaCl2, 0.5% Tritton X-100, pH 7.4) containing 30% % DMSO (v/vv) was incubatted P for 1 h at 37 °°C. The disulfiide bond of 1 was w witth 1 mM TCEP redduced by TCEP P, initiating thee condensationn reaction to yieeld thee cyclized dim mer (i.e., 1-D) which instantlly self-assemblled into the nanopaarticles 1-NPss. Compared with the singgle queenched fluoreescence emisssion at 521 nm of 1, tthe fluuorescence inteensity of 1-NPs dispersion fuurther droppedd to 1/22.8 of its originnal (Figure 2A A), suggesting the efficient duual queenching of thee fluorescencee after nanopaarticle formatioon. Forrmation of 1-N NPs was testiffied by the obvvious increase of thee absorbance aat 500-700 nm m (due to the liight scattering of parrticles) on the UV-Vis specttra (Figure S13). Transmissiion eleectron microsccopy (TEM) image clearlly indicated tthe forrmation of 1-N NPs with an avverage diameterr of 130.3  133.4 nm m (Figure 2B and Figure S S14). High perrformance liquuid chrromatography (HPLC) and electrospray ionization (ESI) maass spectroscoppic analyses inndicated that 1 at retention tim me of 16.7 min was completely connverted to 1-D at retention tim me of 24.5 min (Figuures S15-S16).

Figgure 2. (A) Fluoorescence specttra of 25 μM 1 (black) ( and 25 μ μM 1 incubated i with 1 mM TCEP P at 37 °C for 1 h (i.e., 1-N NPs disspersion) (red) in furin bufferr. Excitation: 465 nm. (B) TE EM imaage of above 1--NPs dispersionn.

Figure 1. Schematic S illuustrations of reduction-conttrolled condensation of 1 to sellf-assemble innto dual quenched

T Then we testiffied the furin--instructed fluoorescence “TuurnOnn” of dual quennched 1-NPs ddispersion and single quenchhed 1-P P. 12.5 μM M 1-NPs disspersion weree obtained by cenntrifugation off above reactioon mixture annd redispersed in sam me volume of ffurin buffer conntaining 10% D DMSO. 25 μM M 1P solution was obtained by dissolved 1-P P in furin bufffer DMSO. As shhown in Figuree 3, without furrin, conntaining 10% D thee background ffluorescence inntensity of the ddual quenchedd 1NP Ps dispersion aat 521 nm (starrred spectrum inn Figure 3A) w was aboout 10-fold loower than thatt of the singlle quenched 11-P sollution (starred spectrum in Fiigure 3B). Afteer incubated with w 0.11 nmol·U-1 furiin for 8 h at 377 °C, the fluorrescence intenssity of 1-NPs disperssion increased 56-fold (Figuure 3A) while tthe Figure 3B). Wee thus concludded thaat of 1-P increeased 5-fold (F thaat our dual queenched 1-NPs had 11-fold more m S/N contrrast thaan that of singgle quenched 11-P for in vitroo furin detectioon.


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To chemicallyy validate aboove fluorescennce “Turn-On”” was induced by furrin cleavage as schematically exampled in Figure F 1B and Figuree S17, we injeected above 1--P reaction miixture into a HPLC syystem for analyysis. As shownn in Figure S188, two new peaks at rretention time of 20.7 min and a 21.3 min w whose masses were reespectively ideentical to thosee of FITC-GR RVRR and GGK(DA ABCYL)G (i.ee., the enzymaatic products oof 1-P after furin cleaavage) (Figures S19-S20), apppeared on the trace of 1-P reactionn mixture. To exactly calcullate the percenntages of furin cleavvage and quenching efficienccy of above 11-NPs dispersion annd 1-P soluttion, we firrstly obtainedd the fluorescence eemission specctra of FITC-GRVRR in furin buffer at diffe ferent concentrrations to estaablish a calibrration curve. As show wn in Figure S S21, a linear reelationship bettween fluorescence inntensity at 5211 nm and conccentration of F FITCGRVRR (Y = 607.1 * X + 63.72, 6 R2 = 0.99) was obtaineed. By fitting this caliibration curve,, we calculatedd that about 111.78% of 1-NPs and about a 8.76% off 1-P were cleaaved by furin after a 8 3 h incubation. Using the forrmula we proposed before,30 the ficiency of 1-N NPs was calcullated to be 99..78%, quenching effi which was biggger than thatt 97.88% of 11-P. These datta not only proved thhat dual quenchhed 1-NPs havve higher quennching efficiency thann single quencched 1-P, but also indicatedd that furin prefers 11-NPs to 1-P fo for cleavage. Kinetic K study ecchoed that the Kcat/K KM value of fuurin towards 1-NPs (0.057 μ μM-1• -1 -1 -1 min ) was aboout 3-fold of thhat towards 1-P P (0.020 μM •min • ) (Figure S22) and the Kcat/KM values in this workk are comparable too those of receently reported furin probes (Table S1).

B Before applyiing 1 for im maging intraceellular furin, we stuudied its stabiliity and specifiicity. Cellular redox status is a preecise balance bbetween the levvels of reactive oxygen speccies (RO OS) (e.g., O2•−, H2O2, •OH) annd reducing sppecies (e.g., GS SH, asccorbate acid).31 HPLC analyyses indicated that our probee 1 hadd excellent staability to 4-foold different R ROS (O2•−, H2O2, − • 1 ClO O , OH, O2) at a 37 °C in furiin buffer for 2 h (Figure S23A A). Specificity of 1 to GSH among a rangge of biological subbstances was ttested. As show wn in Figure S23B, negligibble chaanges in the fl fluorescence inntensity 1 weree observed in tthe preesence of the bbiothiols (Cys, Hcy) except G GSH, ROS (O O2•−, − 1 • H2O2, ClO , O2, OH), or redducing species (ascorbate aciid), sugggesting the exxcellent specificcity of 1 towarrds GSH. A After in vitro iinvestigation, w we then appliedd 1 and 1-P (both weere single quencched) for fluoreescence imagingg of furin activvity in living cells. To overcome thhe interference from intracelluular Cyys and warrant tthe condensationn of 1 for the seelf-assembly off 1NP Ps inside cells, we used 50 μ μM f (i.e., Cyss(StBu)-Lys-CB BT, Schheme S2) to cco-incubate witth 20 μM 1 foor cell imagingg.22 Before that, cytottoxicity of 1 toogether with 500 μM f, or 1-P on The furrin-overexpressiing MDA-MB--468 cells was investigated. T Alaamar Blue assaay indicated thhat more than 90% of the ceells surrvived up to 12 h at the concenntration up to 800 μM of 1 togethher witth 50 μM f, or 880 μM 1-P (Figgure S24), sugggesting that 20 μ μM eithher 1 or 1-P is safe for cell im maging. Time-coourse fluorescennce imaaging of 20 μM M 1 together with w 50 μM f iindicated that, the fluorescence of MDA-MB-468 M cells graduallly turned on aand reaached its plateauu after 2 h (Figuures S25-S26), ssuggesting that the fasst uptake and coondensation of 1 and gradual ccleavage of 1-N NPs by the intracellulaar furin. Then w we chose the inccubation time of o 2 fo the followingg experiments. A As shown in topp row of Figuree 4, h for MD DA-MB-468 cells incubated w with 20 μM 1 toggether with 50 μ μM f showed bright ggreen fluoresceence which welll overlapped w with thee red fluorescennce of furin stainning, suggestingg that 1-NPs w were cleeaved by intraceellular furin. In ccontrast, when tthe cells were prep inccubated with 1 mM furin inhibbitor II (H-(D)A Arg-Arg-Arg-A ArgArgg-Arg-NH2) foor 30 min, theeir green fluorrescence intenssity deccreased by aboout 25-fold (topp middle row oof Figure 4). T This ressult indicated thhat our dual queenched probe 1 could be appllied to detect intracelllular furin acttivity with 25--fold fluorescennce conntrast. When cells were incubbated with 20 μM 1-P (bottom midddle row of Figgure 4), their greeen fluorescence intensity (37.99  4.44) was smaller tthan that of cellls incubated witth 1 (46.6  5.22 in topp row) (Figure S27) which miight due to thee smaller cleavaage perrcentage of 1-P P than that of 1-NPs by furinn. When the ceells weere pre-incubateed with 1 mM furin fu inhibitor III for 30 min, th heir

greeen fluorescencce intensity deecrease by abouut 4-fold (bottoom row w of Figure 4). This result inddicated that thee single quenchhed proobe 1-P could bbe applied to ddetect intracelluular furin activvity witth 4-fold fluuorescence coontrast. Takenn together, we conncluded that, aalthough both 1 and 1-P were single quenched, com mpared with 1--P, our “smart”” dual quenching strategy andd 1 couuld additionallly enhance thee S/N contrastt by 6.3-fold for moore sensitive deetection of intraacellular furin. Figure 3. Tim me-course fluorrescent spectra of 12.5 μM 11-NPs dispersion (A) or 25 μM 1-P P (B) incubatedd with 0.1 nm mol·U-1 furin in furin buuffer for 0.5 h, 1 h, 2h, 3 h, 4 hh, 5 h, 6 h, 7 h, and 8 h at 37 °C. Exccitation: 465 nm m.



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otes No The authors declarre no competingg financial interrest.

AC CKNOWLED DGMENT This work was suupported by Coollaborative Innnovation Centerr of Suzzhou Nano Science and Techhnology, the Major M program of Deevelopment Fouundation of Heefei Center for Physical Sciennce andd Technology (2016FXZY0006), Ministry of Science aand Tecchnology of C China (2016YF FA0400904), aand the National Naatural Science Foundation off China (Grantts 21725505 aand 216675145).

RE EFERENCES

Figure 4. Connfocal fluoresceence and overlaay images of M MDAMB-468 cells inncubated with 220 μM 1 togetheer with 50 μM f for 2 h (top row), pree-incubated withh 1 mM furin innhibitor II for 30 min then 20 μM 1 ttogether with 500 μM f for 2 h (top middle row w), 20 μM 1-P for 2 h (bottom midddle row), pre-inncubated with 1 mM furin inhibitor III for 30 min thhen 20 μM 1-P for 2 h (bottom m row) in serum-free DMEM D at 37 °C C. Blue is Hoechhst 33342 stainning of nucleus, greeen is from FITC in 1 or 1-P, reed is immunofluoresscence staining oof furin. Scale bbar: 10 μm.

In summaryy, by employinng a click conndensation reaaction and rational design of a single quencched probe 11, we “ dual qquenching strattegy to enhancce the developed a “smart” detection sensitivity of inntracellular furrin. In vitro tests validated that,, at physiologiical condition and pH 7.4, 1 was subjected to reduction-conttrolled condennsation reactioon to form 1-NPs aand its fluoresccence intensityy at 521 nm fuurther dropped to 1/22.8 of its originnal. Upon furinn cleavage, thee dual quenched 1-NP Ps had fluoresccence “Turn-O On” contrast 11 folds more than thatt of single quennched control probe p 1-P. Livve cell imaging resultts indicated thhat, although tthe fluorescennce of both 1 and 1-P P were single quenched, witth our “smart”” dual quenching sttrategy, 1 shhowed fluorescence “Turnn-On” contrast 6.3-foold of that of 1--P for furin im maging. We envvision that, by replaacing the RRV VR substrate w with other enzzymecleavable oness, our versatilee “smart” dual quenching strrategy could be easilyy adjusted for tthe detection (oor imaging) off other intracellular ennzymes’ activitty with enhancced sensitivity.

ASSOCIATE ED CONTEN NT Supporting In nformation General methoods; Synthesess and characterizations of 1--P, 1, and FITC-GR RVRR; Schem me S1-S3; Figgure S1-S27; Table S1-S3. This m material is availaable free of chaarge via the Intternet at http://pubs.aacs.org.

AUTHOR IN NFORMATIO ON Corresponding Author *E-mail: [email protected].

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