Gold nanoparticles and uorescently-labelled DNA as aplatform for biological sensing

Amelie Heuer-Jungemann,a Pascal K. Harimech,a Tom Brownbc

and Antonios G. Kanaras*ac

In the past decade gold nanoparticlenucleic acid conjugates became progressively important for

biomedical applications. Fluorophores attached to nucleic acidgold nanoparticle conjugates have

opened up a new era of biologic

invention of the so-called nano

endocellular targets such as mRNA

discuss the current progress in th

cellular uptake and cytotoxicity.

detect targets within the cellular environment in real time. In and highly sensitive endocellular sensors has become apparent.

AriHb

aPhysics and Astronomy, Faculty of Physica

Southampton, Southampton, SO17 1BJ, UK.bChemistry, Faculty of Environmental

Southampton, Southampton, SO17 1BJ, UKcInstitute for Life sciences, University of Sou

Cite this: DOI: 10.1039/c3nr03707j

Received 18th July 2013Accepted 19th August 2013

DOI: 10.1039/c3nr03707j

www.rsc.org/nanoscale

Nanoscale

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View Article OnlineView Journalat the third year of her PhD,

Psrpb

This journal is The Royal Society ofAnswering this requirement, scientists have developed a noveldetection system based on uorescent DNA attached to goldnanoparticles.2836 Oligonucleotidenanoparticle conjugates areincreasingly employed in various types of sensing applicationsespecially the ones involving endocellular sensing.2931,3741 This

melie Heuer-Jungemanneceived her MChem in Chem-stry with Biochemistry fromeriot-Watt University, Edin-urgh in 2011. She is currently

hysics and Astronomy, Univer-

Pascal Harimech obtained hisDiploma degree in Physics fromthe Philipps-University of Mar-burg in Germany in 2012. Pascalis currently at the second year ofhis PhD, Physics and Astronomy,University of Southampton. His

l Sciences and Engineering, University of

E-mail: A.Kanaras@soton.ac.uk

and Natural Sciences, University of

thampton, Southampton, SO171BJ, UK1. Introduction

The cell is one of the most complex biological environmentscontaining a vast number of dierent biomolecules withmultiple biological roles. Being able to detect these biomole-cules with great selectivity is of high importance in order tounderstand biological processes, disease progression and toformulate and monitor specic treatments. Common sensingmethods for the detection of biologically important mole-cules15 are frequently based on platforms of nucleic acids dueto their inherent property to selectively bind a complementarytarget.615 However, most of these methods are not designed toity of Southampton. Heresearch is focused on nano-articleDNA conjugates foriomedical applications.

Chemistry 2013al sensing. The most promising advancement in this eld was the

-are systems. These systems are capable of detecting specic

s, microRNAs or small molecules in real time. In this minireview, we

e eld of DNAnanoparticles as sensors, their properties, stability,

order to advance the eld towards detection in living cells,uorescent nucleic acid probes such as hairpin-based molec-ular beacons have been developed (Fig. 1).1620 Although theseprobes are capable of detecting targets in living cells,1619,21 theirdelivery into cells is extremely challenging. To assist nucleicacid-based probes to enter cells, the use of co-carriers isrequired,22,23 which can exhibit signicant cytotoxicity.2426 Butthis is not the only diculty with nucleic acid-based sensors.Once delivered into the cell, free nucleic acids are inherentlyprone to digestion by nucleases.27

On account of the aforementioned problems with conven-tional detection systems, the urgent need for new types of stableresearch focuses on chiralplasmonics and optical proper-ties of DNAgold nanoparticleassemblies.

Nanoscale

factors such as (a) the emission wavelength of the uorophore,(b) the orientation of the uorophore relative to the surface ofthe particle, (c) the distance from the nanoparticle surface aswell as the morphological characteristics of the particle (whichare strongly related to the characteristics of the plasmoniceld).35,4244 Libchaber and co-workers were among the rstgroups to extensively study the quenching eciency of 1.4 nmgold nanoparticles with respect to dierent dyes (Rhodamine6G, Fluorescein, Texas Red, Cy5).34 Their design involved a u-orescently tagged hairpin loop developed as a sensor for single-mismatches in DNA and functioned in a similar way to amolecular beacon.34,45 The oligonucleotide anchored to the goldnanoparticle was functionalized with a uorophore, which, dueto the close proximity to the gold nanoparticle was completelyquenched.42,43 Upon target binding, the loop would open anduorescence would be restored due to the increase in thedistance of the uorophore from the gold nanoparticle. In theirstudies it was found that all dyes were quenched more e-ciently than when DABCYL, a quencher oen used in conven-tional molecular beacons, was used.34 Maxwell et al. employed asimilar strategy using 2.5 nm AuNPs.35 Due to their excellentquenching abilities, small particles are oen used for these

Fig. 1 Principle of molecular beacons. A hairpin DNA is functionalized with auorophore and a quencher. Upon target binding the hairpin is opened up, thedistance between quencher and uorophore increases and a uorescent signalcan be observed.

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View Article Onlineminireview discusses the dierent types of gold nanoparticleoligonucleotide sensors reported to date, their design, emergingapplications and their basic characteristics such as biologicalstability, functionality, cellular uptake and cytotoxicity.

2. Design of oligonucleotidegoldnanoparticle sensors

The general concept of uorophore-tagged nucleic acidgoldnanoparticle conjugates as biological sensors relies on theuorescence quenching ability of gold nanoparticles. A uo-rophore placed within a few nanometres of a metal nanoparticleexhibiting a strong plasmon eld, experiences enhancing orquenching eects due to its interactions with the plasmoniceld.42 The degree of enhancing or quenching depends onmanyTom Brown was appointed to alectureship in Chemistry atEdinburgh University in 1985where he was promoted toProfessor of Nucleic Acid Chem-istry. He then moved to South-ampton University in 1995 andwill join Oxford University in2013. Tom's research interestscentre on nucleic acid chemistry,structure, DNA sequence recog-nition and their applications inbiology and medicine.

Nanoscalepurposes.35 However, if a detection system, based on a similarmodel was to be adopted for in vitro applications, small goldnanoparticles may not be suitable because they have shown toexhibit signicant cytotoxicity. On the contrary, larger particles(up to 50 nm) have shown good cellular uptake and negligiblecytotoxicity.46,47Due to the ease of synthesis and low cytotoxicity,1315 nm AuNPs are most widely employed in cellular appli-cations, but in some cases larger (up to 20 nm) AuNPs are useddue to a higher oligonucleotide loading capability.4850 Based onthis hypothesis, Mirkin and co-workers developed a sensor formRNA detection in living cells (Fig. 2A).36 Their so-called nano-are sensor consisted of oligonucleotidefunctionalized goldnanoparticles hybridised to a are/reporter stranda short

Antonios Kanaras received hisdiploma in Chemistry from theUniversity of Crete, Greece in1996. Aer a Master's degree inBioinorganic Chemistry from theUniversity of Ioannina, Greece,he received a PhD in Chemistryfrom the University of Liverpoolin 2004. He was a postdoctoralscientist at the University ofCalifornia, Berkeley working onthe synthesis and energy appli-cations of semiconductor nano-

particles. In October 2007 he became an Assistant Professor inPhysics and Astronomy, University of Southampton and in March2012 he was promoted to Associate Professor. His research focuseson synthesis, functionalization and self-assembly of colloidalnanoparticles as well as their applications in Biomedical andPhysical Sciences.This journal is The Royal Society of Chemistry 2013

inmviio

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View Article Onlineoligonucleotide strand functionalised with a uorophore. Dueto the quenching ability of the gold nanoparticle, detectableuorescence from the reporter strand is negligible. Upon targetbinding, the reporter strand is released through competitivehybridisation and a uorescence signal correlating directly withthe relative amounts of target can be observed.36 Followingthese initial results, the sensor was then advanced further forsimultaneous mRNA detection and regulation.29 For thoseexperiments, HeLa cells were treated with a high concentration

Fig. 2 (A) Schematic illustration of nano-ares capable of entering cells and detectcompetitive hybridisation leading to a detectable increase in uorescence. (B) AptaSKBR3 and non-survivin expressing C166 cells treatedwith survivin nano-ares (Surare displayed accordingly below each image. (A and C) re-printed with permisspermission from ref. 30. Copyright 2009, American Chemical Society.of survivin mRNA detection nano-ares (5 nM). Mirkin and co-workers were able to detect the survivin target in living cellculture and additionally a 92 4% depletion in survivin mRNAwas observed, implying the gene regulatory possibilities of theirnano-ares.29 Some years later the same group advanced thiswork and developed a multiplexed nano-are system for thesimultaneous detection of two dierent targets.28 Li et al. thenadapted this approach and developed a multiplexed probecapable of detecting up to three dierent mRNA targets simul-taneously.32 These multiplexed probes were shown to be espe-cially useful in the eld of cancer diagnosis.28,32

Cancer diagnosis

Cancer related oligonucleotide sequences have been investi-gated especially in the context of nano-are probes as thistechnology is a promising candidate for cancer diagnosis andtreatment.51

As described in the previous paragraph, Mirkin and co-workers developed a nano-are designed for the detection of thesurvivin mRNA transcript. The survivin protein is expressed inhigh numbers in various human cancers.52,53 Survivin inhibitsthe apoptosis pathway of the cell while regulating prolifera-tion.51,54 To test the nanoare probe approach, cells from the

This journal is The Royal Society of Chemistry 2013SKBR3 breast cancer cell line, which are known to express highlevels of survivin, were treated with nano-ares containing fullycomplementary oligonucleotide strands to the survivin tran-script.36 Flow cytometry analysis yielded a 2.5-fold higher uo-rescence signal than that of non-complementary nano-ares. Incontrast, nanoares containing complementary and mis-matched DNA inmouse endothelial cells from the C166 cell linenot expressing the survivin transcript did not show any uo-rescence (Fig. 2C).28,36

g a specic mRNA target. UponmRNA binding the are strand is released througher nano-ares for the detection of intracellular levels of ATP. (C) Survivin expressingvin) and non-complementary nano-ares (Control). Additional ow cytometry datan from ref. 36. Copyright 2007, American Chemical Society. (B) re-printed withFollowing this example, Gu and co-workers developed asensor for the detection of the STAT5B transcript, an importantprotein involved in tumour proliferation andmetastasis.31 Theirapproach, as opposed to the one proposed by Mirkin and co-workers, did not involve a separate reporter strand, but rather atwo in one system in which the uorophore was attached to ahairpin oligonucleotide (Fig. 3). Upon target binding the hairpinwould open and a uorescence signal could be observed.

Likewise, Tu et al. created a system for the detection ofmicroRNA.55 The potential advantage of this strategy is thelocalised signal, as the uorophore is still anchored to the gold

Fig. 3 A molecular-beacon type DNA sensor based on uorophore-tagged,hairpin oligonucleotide functionalised gold nanoparticles.

Nanoscale

nanoparticle instead of being released into the cell's cytoplasm.In this way Gu et al. showed that when their sensors wereincubated with MCF-7 human breast cancer and C6 mouseglioma cell lines, only the human STATB5 expressing MCF-7cells showed a high uorescent signal. Using ow cytometrythey showed that a 4.7-fold increase in uorescence was ach-ieved.31 The authors showed that their probe made it possible tovisualize STAT5B gene expression in real time, which allowedrapid identication of tumour progression stages as well asevaluation of anti-cancer treatment outcomes.

Tang and co-workers32 adapted a similar approach to Mir-kin's nano-are in order to monitor tumour progression, butinstead of just one or two, they studied three dierent mRNAtargets. Detecting multiple targets simultaneously is of greatimportance in order to properly determine the stage a tumourhas reached. It has been shown that cancer cells can expressseveral mRNA sequences at varying levels depending on thestage of the tumour. Therefore a multiplexed probe for mRNAsassociated with cancer development (C-myc, TK1 and Gal-NAc-T), was designed by the authors. These sensors wereintroduced to simultaneously investigate the three mRNAsequences in both, MCF-7 human breast cancer cells and MCF-10A normal human breast cells as well as in HepG2 human livercancer cells and HL-7702 normal liver cells. Cells were incu-

been measured. This result indicates the importance of multi-plexed sensors as being more reliable against false positiveresults. To account for dierent amounts of mRNA expressed inthe cells, the group incubated them with the drugs tamoxifenand b-estradiol, which inhibit or promote the expression ofTK1, respectively.56,57 They found that the dierent levels of TK1in the cells could be distinguished allowing to estimate therelative amounts and therefore to evaluate the state of tumourprogression.

Ion and small molecule detection

The importance of detecting biological molecules such asmRNA or microRNA has been discussed in the previous para-graphs; however the detection of other small molecule targetsand ions especially heavy metal ions is equally importantand has been investigated. In the same way that general single-stranded DNA has great specicity for its complementary targetsequence, aptamer DNA is widely known for its target specicitywith respect to small molecules and ions. Building on thisknowledge, aptamer nano-ares were developed by Mirkin andco-workers (Fig. 2B) as well as Chung et al.30,33 These nano-areswere capable of detecting intracellular levels of ATP or heavymetal ions in human serum.30,33 In these probes target binding

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View Article Onlinebated with the multiplexed nano-ares and analysed usingconfocal laser scanning microscopy (Fig. 4). While in the breastcancer cells all three mRNAs are present in a high number,there was no detectable uorescence in healthy breast cells.Likewise, cancerous liver cells showed uorescence of all threedyes indicating expression of all three mRNA. However, inhealthy liver cells GalNAc-T was also found. Even though thesignal in healthy cells was weaker, these cells might have beencharacterised as cancerous if only the GalNAc-T sequence had

Fig. 4 Multicolour nanoprobe for the detection of c-myc, TK1 and GalNac-T mRmRNAs in four dierent cell types. Re-printed with permission from ref. 32. CopyrNanoscaleAs. Confocal microscopy images show the dierent expression levels of all threeht 2012, Wiley.induces a conformational change in the DNA resulting inreporter strand release and the formation of a folded DNAstructure such as a hairpin or a G-quadruplex.30,33

Besides aptamer DNA, a new approach using hollow poly-electrolyte multilayer capsules has been shown to be a prom-ising candidate for the detection of ions in vitro and in vivo.58

Recently, a multiplexed probe to detect potassium and sodiumions and the pH simultaneously using four dierent uo-rophores was presented.59This journal is The Royal Society of Chemistry 2013

3. Characteristics of oligonucleotidegoldnanoparticle sensors

In order for the oligonucleotidegold nanoparticle sensors to besuitable for biological applications, it is of great importance forthem to be very stable in cellular environments, display hightarget specicity and low cytotoxicity. Thus various tests havebeen undertaken to investigate specic properties of dierenttypes of these nano-sensors.

Stability

The nano-ares were exposed to various conditions commonwithin a cellular environment, such as dierent enzymes as wellas thiol-containing molecules such as glutathione, which couldresult in displacement of the oligonucleotide strand from thegold nanoparticle. Fig. 5 shows that both digestion by nucleasesand displacement of the sensing strand from the gold nano-particle surface are negligible within hours, with degradation

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View Article OnlineFig. 5 (A) Fluorescence curve of nanoprobes in the presence (grey) and absence(black) of DNAse I. (B) Fluorescent microscopy images of dual-uorophorelabelled DNAAuNPs (30-Cy3 and 50-Cy5.5) incubated with C166-EGFP cells for 48h. Only Cy5.5 uorescence can be observed (upper left), whilst Cy3 uorescence isnot visible (upper right), indicating that the thiolated oligonucleotides remainattached to the particle. (A) re-printed with permission from ref. 32. Copyright2012, Wiley. (B) from ref. 77. Re-printed with permission from AAAS, Copyright2006.charge of the DNA, could be the reason for less ecient enzy-matic degradation. Further numerical calculations by Olvera dela Cruz et al. supported this hypothesis.60

Functionality

Apart from the reports related to the nano-are biologicalstability, the target specicity of these sensors was also inten-sively investigated. Thus, studies into are release and uo-rescence signal detection caused by non-target specicpathways have been carried out. Fig. 6A and B show the resultsof dierent detection systems treated with fully complementaryrates of 0.10.275 nmol min1 4.5 fold lower than for purelyoligonucleotide-based molecular beacons. This proved that thenano-are is a highly stable system under stringent condi-tions.32,36Mirkin et al. investigated the unexpected high stabilityof oligonucleotideAuNP conjugates to enzymatic degradation a common problem in sensors made from oligonucleotides.According to their study, the high local sodium ion concentra-tion around the dense DNA corona, arising from the negativeThis journal is The Royal Society of Chemistry 2013Cellular uptake

The nal focus should be on the interaction of these systemswith cells and the determination of their cytotoxicity. Variousgroups have explored the uptake mechanism of functionalisedgold nanoparticles. Our group amongst many others hasinvestigated the eects of size, shape and functionality ofnanoparticles on cellular uptake.47,48,6173

It is widely known that positively charged species enter cellsmuch more readily than negatively charged ones, due to theirelectrostatic interactions with the negatively charged lipidmembranes.27 Thus, in many studies, in order to introducenucleic acid-based systems into cells, complexation with posi-tively charged co-carriers was carried out.22,23,25 However, in thecase of nano-ares, it has been shown that DNAAuNP conju-gates, although highly negatively charged, are taken up by cellsin large numbers48 without the aid of a co-carrier.27,48 Investi-gating this phenomenon, Mirkin and co-workers rst showedthat the oligonucleotide loading plays an important role inDNAAuNP uptake. For surface loadings of more than 18 pmolcm2, cellular internalisation of a very high number of DNAAuNPs per cell was possible.27,48

Since this discovery, studies on the uptake mechanism ofDNAAuNP conjugates have received signicant attention, yetto date the mystery is still not completely solved. It was shownthat within the cellular environment, the hydrodynamic radiusof DNAAuNPs was greatly increased and a change in chargecould be observed.48 One hypothesis is that these changes weretargets and mismatched targets. It is evident that even a singlemismatch within a target strand causes no more than 50%release of reporter strand.

In the case of aptamer nano-ares, the target specicity isequally high as would be expected. The heavy metal aptamer-based sensors were highly specic towards Hg2+ and Pb2+

ions and could tolerate other ions contained within humanserum. A similar result was observed with aptamer nano-aresselective for adenosine triphosphate (ATP). While an immediateresponse for physiological concentrations of ATP was obtained,the aptamer ares did not sense similar molecules such asguanidine triphosphate (GTP), cytosine triphosphate (CTP) oruridine triphosphate (UTP).30 All authors concluded that fullycomplementary targets provide a signicantly higher signalthan single-mismatched. The uorescence intensity of Mirkin'snano-ares showed a 3.8-fold increase against the backgroundaer target addition.36 Tang's multiplexed nano-sensorsreached a 4.4 to 5.9-fold increase of uorescence for thedierent sequences employed.32

Additional to their high target specicity, a further advan-tage of nano-are style sensors is their detection speed. Fig. 6Cshows a time-dependent study of reporter strand release uponaddition of the relevant target. Already aer 5 min, all of thereporter strands were released and a high increase in uores-cence was observed. Fig. 6D indicates the high selectivity ofaptamer nano-ares towards the desired heavy metal iontargets. All the above studies established the nano-are systemto be a highly selective sensor with very fast response rates.Nanoscale

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View Article Onlinecaused by the adsorption of cellular proteins, which assistedinternalization.48 However, further investigations demonstratedthat adsorption of common cellular proteins such as bovineserum albumin (BSA) and transferrin resulted in reducedcellular uptake, which contradicted previous observations.74

Latest studies claimed that scavenger receptors receptorsinvolved in the recognition and uptake of anionic macromole-cules75 mediate the uptake of oligonucleotidefunctionalizedgold nanoparticles (Fig. 7). It was discussed that oligonucleo-

Fig. 6 (A) Fluorescence spectra of nano-ares (green curve) treated with a non-cFluorescence spectra of nano-ares treated with targets containing up to four micontrol target (red). (D) Aptamer nano-ares for the detection of Hg2+ and Pb2+ ionfrom ref. 36. Copyright 2007, American Chemical Society. (B and C) re-printed withfrom ref. 33 with permission from Elsevier, Copyright 2012.tides mimic the complex structure of Poly I, a well-knownbinding ligand for scavenger receptors, resulting in internali-zation. Recently Mirkin and co-workers further showed thatclass A scavenger receptors as well as lipid-ras are involved inthe endocytosis mechanism of gold nanoparticleDNA conju-gates.76 All these reports have shed more light on the uptakemechanism of DNAgold nanoparticle conjugates, howeverdetails of the exact mechanism of cellular internalizationremains a subject of intensive investigation.

Fig. 7 Schematic illustration of the proposed cellular uptake mechanism ofDNAAuNP conjugates. In media containing serum proteins, coating of DNAAuNPs with these proteins reduces cellular uptake. In the absence of serumproteins, interactions between DNAAuNPs and scavenger receptors are aidedand uptake is increased. Re-printed with permission from ref. 74. Copyright 2010,American Chemical Society.

NanoscaleCytotoxicity

Finally, it is of utmost importance for the nano-are sensors toexhibit as little cytotoxicity as possible. Several studies showthat there is a strong indication that gold nanoparticles func-tionalized with oligonucleotides are not cytotoxic. Mirkin et al.did not observe any toxicity of the nano-ares in cell antisenseexperiments.77 In another study, Tang et al. conducted an MTTassay78 to assess the cell viability with their multiplexed nano-

32

plementary target (blue curve) and a fully complementary target (red curve). (B)atches. (C) Time scan of Survivin nano-ares treated with survivin (black) and ain human serum display excellent target specicity. (A) re-printed with permissionermission from ref. 29. Copyright 2009, American Chemical Society. (D) Re-printedares. The authors reported that within concentrations ofaround 5 nM and an incubation time of up to 48 hours, the cellviability was always greater than 85%. Likewise, an MTT assaycarried out by Gu and co-workers revealed that the viability ofMCF-5 and C6 mouse glioma cell lines was not aected by thetreatment with their nano-sensor.31 It can therefore beconcluded that the discussed oligonucleotideAuNP baseddetection systems exhibit negligible cytotoxicity. Along withtheir high stability, great target specicity and good cellularuptake these sensors are thus ideal agents for biologicalapplications.

4. Summary and outlook

The rst demonstration of oligonucleotidefunctionalised goldnanoparticles about two decades ago created a cornerstone formodern bionanotechnology. High impact research has beenconducted in the direction of biological sensing by exploitingthe unique recognition properties of oligonucleotides. At thesame time manifold surface functionalization molecules suchas peptides, antibodies or transfection agents, to promote theDNAgold nanoparticle uptake in living cells, were utilized. Thesurprising discovery that gold nanoparticles densely coveredwith oligonucleotides readily enter living cells formed a new

This journal is The Royal Society of Chemistry 2013

more highly advanced multiplexed systems will be designed

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Notes and references

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11 P. O. Brown and D. Botstein, Nat. Genet., 1999, 21, 3337.12 J. J. Chen, Pharmacogenomics, 2007, 8, 473482.milestone in the area of nano-sensing and nano-therapeutics.Since then, dierent oligonucleotideAuNP sensing systemshave emerged and have even recently been commercialised byAuraSense andMilliPore as detection agents for microRNAs inliving cells. The inherent advantages of nano-are style systemsare their ease of cellular uptake, high stability in a cellularenvironment, greatly increased resistance towards degradationby nucleases, high target specicity and low background uo-rescence. They also exhibit signicantly lower innate immuneresponse than comparable DNA systems delivered usingcommercial transfection agents.36,79,80 This makes these systemshighly valuable for biological sensing applications and superiorto many other detection methods.2833

Recently, a new approach towards nano-are-like sensorswithout a gold nanoparticle support has been provided by Tanet al. who synthesised molecular beacon micelle ares(MBMFs).81 This sensory system consist of a hairpin DNAmodied at both the 30 and the 50 end with a uorophore and aquencher respectively.45 The oligonucleotide was furthermodied with a diacyllipid phosphoramidite82 bearing two longhydrophobic C18-hydrocarbon tails. With a critical micelleconcentration below 10 nM, these molecules readily formmicelles in aqueous solution with the hairpin pointing to theoutside and therefore being able to be reached by target mRNA.Further investigations into how these novel systems compare tonanoparticle-based detection systems will be required.

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