multicolor fish probes using qd-dna conjugates - weiss, cel biochem biophys 2006

ORIGINAL ARTICLE

Copyright 2006 by Humana Press Inc.All rights of any nature whatsoever reserved.1085-9195/(Online)1559-0283/06/45:5970/$30.00

Cell Biochemistry and Biophysics 59 Volume 45, 2006

INTRODUCTION

Fluorescence in situ hybridization (FISH) of nucleicacid-labeled probes provides a direct visualization of thespatial location of specific DNA or RNA sequences at aparticular cellular or chromosomal site and in tissue sec-tions. Such morphological and topological informationabout the localization of the target sequence has beeninvaluable for both fundamental and applied research,including studies of gene organization, function and reg-ulation during embryo development, detection of viralinfections, and chromosome rearrangements associatedwith genetic disorders (1). FISH has the capacity tosimultaneously visualize different targets in multiple,distinct colors. However, because of the spectral overlapof commonly used organic fluorophores, the number ofprobes identifiable on the basis of a unique fluorescencecolor is typically limited. Also, the multiple wavelengths

required to excite polychromatic specimens further intro-duce chromatic aberrations that can reduce the reliabilityof multicolor fluorescent probes co-localization measure-ments. Finally, rapid photobleaching of most organicdyes may further compromise imaging of the sample.

Nano-sized semiconductor crystals known as quan-tum dots (QDs) have emerged as new fluorescent labelsthat possess several optical properties that could funda-mentally overcome these limitations (2). First, QDsemission wavelength can be fine-tuned anywhere fromthe ultraviolet (UV) to the infrared by means of materialcomposition and size from quantum confinementeffects. Second, QDs have very narrow and symmetricemission bands (2530 nm full width at half maximum)allowing multiple colors of QDs to be used with mini-mal spectral overlap. Third, QDs have a wide absorp-tion band therefore allowing all of the possible QDcolors to be simultaneously excited with a single nar-row-band excitation source and distinguished in a sin-gle exposure, thus eliminating chromatic aberrations.Fourth, QDs are very photostable and emit many morephotons per particle compared to dye molecules.

Single-Step Multicolor Fluorescence In Situ Hybridization Using Semiconductor Quantum DotDNA Conjugates

Laurent A. Bentolila1,2,* and Shimon Weiss1,2,3

1Department of Chemistry and Biochemistry, 2California NanoSystems Institute, and 3Department of Physiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1569

Abstract

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences usingnovel quantum dot-based fluorescence in situ hybridization (FISH) probes (QDFISH). Short DNA oligonu-cleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QDFISHprobes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting ofdense chromatin domains with minimal steric hindrances. We further demonstrated that QDs broad absorptionspectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneouslyexcited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus,these results demonstrate that QDFISH probes are very effective in multicolor FISH applications. This workalso documents new possibilities of using QDFISH probes detection down to the single molecule level.

Index Entries: Quantum dots; FISH; DNA; heterochromatin; centromere; fluorescence; imaging; microscopy;hybridization; biomaterial; nanotechnology.

* Author to whom all correspondence and reprint requestsshould be addressed. E-mail: [email protected]

Received: 4/8/05; Revised: 9/1/05; Accepted: 9/14/05

A recent study has shown that the superior in situphotostability of QD-labeled FISH probes comparedwith conventional organic dyes can improve quantita-tion of the FISH signals (3). However, QD usage in FISHhas thus far been limited to single-color detection of sin-gle genetic targets (3,4). Current limitations to multi-plexing include the paucity of robust chemistry tobioconjugate DNA probes directly to QDs (4) and thelack of availability of diverse hapten-functionalizedQDs to allow multicolor secondary detection schemes(3). Finally, it has remained unclear whether the 10 to 20times larger size (compared with the size of conven-tional organic dyes) of the QDs would limit their use inmulticolor FISH applications.

Here, we report the preparation of novel QDFISHprobes based on the direct attachment of short DNAoligonucleotides onto QDs and their use in multicolorFISH analysis. We show in a proof-of-principal experi-ment that two QDDNA probes with different emissionspectra could be used in a one-step hybridization/detection experiment to visualize in situ two subchro-mosomal regions of highly condensed chromatin struc-ture within the centromere. QDs broad excitationspectrum allowed the two different color probes to besimultaneously excited by a single excitation wave-length and distinguished in a single exposure usingstandard far-field optics. These results demonstrate thatQDDNA probes are very effective in multicolor FISHapplications and may offer the opportunity to achievetrue multicolor identification and analysis of chromoso-mal structures at high resolution.

MATERIALS AND METHODS

Cell Culture and FixationThe mouse mast cell line P815 and the human cervical

epithelial cancer cell line HeLa (cat. no. TIB 64 and CCL-2, respectively, American Type Culture Collection,Manassas, VA) were maintained in complete Dulbeccosmodified Eagles medium (Gibco-Invitrogen, Carlsbad,CA) supplemented with 5% fetal calf serum, L-glutamine,and antibiotics. Cultures were kept at 37C in 5% CO2and maintained at 80% confluence. Metaphase spreadswere prepared by hypotonic treatment of Colcemid-treated cell cultures (KaryoMax, Gibco-Invitrogen) andfixed in 3:1.5 (v/v) methanol/glacial acetic acid. Cellswere then dropped on glass cover slips following stan-dard procedures.

ReagentsAll reagents were of analytical grade. Qdot Strep-

tavidin Conjugates 525, 565, 585, 605, and 655 nm werepurchased from Quantum Dot Corporation (Hayward,

CA). Some early lots of 525, 592, 611, and 655 nm QDswere also provided from the same manufacturer forbeta testing.

Preparation of QDDNA ComplexesQDDNA complexes were formed by incubation of

biotinylated DNA oligonucleotides with QDs at variousmolar ratios at room temperature for 30 min. Remainingfree streptavidin sites were further blocked with anexcess of biotin. QDDNA complexes were analyzed byelectrophoresis on a 2% agarose gel in Tris-Borate-EDTA(TBE) 0.5% and characterized by electromobility shiftassay. Single-stranded DNA was counterstained withSYBR Green II (Molecular Probes, Eugene, OR). Themigration patterns of the QDs and the DNA were eachspectrally determined with a MultiImager FX System(Bio-Rad, Hercules, CA) as follow: QDs (532 nm greenlaser excitation, 640-nm band pass (640 BP) red emissionfilter) and SYBR Green DNA (488-nm blue laser excita-tion, 530 BP green emission filter). Titration of the com-plexes were performed for each individual sequences.For most experiments a 1:12 molar ratio of QDDNAwas used. QDDNA complexes were diluted inhybridization buffer (25% deionized formamide, 2Xstandard sodium chloride-sodium citrate [SSC], 200ng/L sheared salmon sperm DNA, 5X Denhardts, 50mM sodium phosphate, pH 7.0, and 1 mM EDTA) at afinal concentration of 1 to 5 ng/L probe and eitherused directly or further purified over a S300 size exclu-sion spin column (Amersham Biosciences, Piscataway,NJ) to remove small amounts of unbound ligands(streptavidin, biotin, and oligonucleotides). The QD-probe mixture was stored at 20C and the fluorescencewas stable for several weeks.

Oligonucleotide SequencesOligonucleotides were synthesized on an Applied

Biosystems 392 DNA/RNA synthesizer (Foster City,CA) or obtained from Invitrogen Corporation (Carlsbad,CA). Biotin or Texas Red was linked to the 5 end of theoligonucleotide through a hexamethylene linker. Mousesequences used in this study are the following: Major -satellites: 5-ATT TAG AAA TGT CCA CTG TAG GAC-3, 5-CCT WCA GTG TGC ATT TCT CAT TTT TC-3;minor satellites: 5-TGA TAT ACA CTG TTC TAC AAATCC CG-3. An irrelevant oligonucleotide: 5-GGG TGTGTC CTG TCG TAG GTA AAT AAC TGA-3 specific toLambda phage DNA was used as a negative hybridiza-tion control (a gift from J. Antelman, Department ofChemistry and Biochemistry, UCLA). Sense and anti-sense oligonucleotides used in the liquid hybridizationassay described in Fig. 1 are: 5-GGG TTA GGG TTAGGG TTA GGG TTA GGG TTA GGG TTA-3 and 5-TAA

60 Bentolila and Weiss

Cell Biochemistry and Biophysics Volume 45, 2006

CCC TAA CCC TAA CCC TAA CCC TAA CCC TAACCC-3, respectively.

In Situ Hybridization ProcedureSlides were pretreated for hybridization by a 0.01%

pepsin digestion in 0.01 M HCl for 5 min at 37C fol-lowed by a short wash in 2X SSC, dehydrated in 70, 90,and 100% graded ethanol series and air-dried. Targetsequences were denatured for 2 min in 70% formamide,2X SSC, pH 7.0, at 70C followed by 70, 90, and 100% ice-cold graded ethanol series and air-dried. Hybridizationof the probe was done by pipetting 14 L of theQDDNA probe hybridization mixture onto the cells.The slides were then baked for 3 min at 85C and incu-bated overnight at 37C in a humidified closed chamber(humidifier is 25% formamide, 2X SSC). Successiveposthybridization washes in 2X SSC (37C for 10 min,

four times), 0.1 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%Tween-20 (room temperature for 5 min, two times) wereused to remove nonspecifically bound and unboundprobe from the cell preparations. Stained cells weremounted on slides in 90% (v/v) glycerol-10% phos-phate-buffered saline with coverslips. QD fluorescencewas stable for several weeks at 4C in the dark, butshowed irreversible time-dependent decay. In controlexperiments, antibody detection of the biotin or digoxi-genin labels was essentially as described by Dirks et al.(5) with three antibody detection steps. Mounting was inDAPI/DABCO: Vectashield (1:1) in 90% glycerol.

Fluorescent Confocal MicroscopySamples were photobrightened with UV and imaged

on a Leica TCS SP2 AOBS confocal microscope (LeicaMicrosystems Inc., Exton, PA) using a 405-nm diode

FISH Using Semiconductor Quantum DotDNA Conjugates 61


Fig. 1. Preparation and characterization of QDDNA complexes used in FISH. (A) Decreasing amounts of streptavidin-coated QD605 (red) incubated with biotinylated DNA oligonucleotides (green fluorescence originating from the SYBRgreen counterstaining) were electrophoresed on a 2% agarose gel in TBE 0.5%. Free DNA (left lane) migrates faster thanQDDNA conjugates (middle lanes) that migrate faster than QDs alone (right lane). Changes in mobility of the QDDNAcomplexes correlate with yellow fluorescence color co-localization. (B) Control experiment with the same DNA sequencebut unbiotinylated. (C) Schematic of liquid-phase hybridization with two color QDs functionalized with combinations ofsense or antisense oligonucleotides. Recognition and hybridization of two self-complementary (S/) sequences shouldresults in the formation of large aggregates. (D) Gel electrophoresis of various combinations of one and two color sense orantisense QDDNA probes after liquid-phase hybridization. Lanes 1 and 10: QDs alone (green 525 nm, red 605 nm, respec-tively), no DNA; Lanes 3, 5, 12, and 14: DNA alone, no QDs. Lanes 2 and 4: green QD525-DNA complexes (sense and anti-sense, respectively); Lanes 11 and 13: red QD605DNA complexes (sense and antisense, respectively); Lanes 6 to 9:hybridization challenges with two colors of sense or antisense QDDNA probes (green and red).

laser (Coherent Inc., Santa Clara, CA) for excitation. Thefluorescence spectrum of each QDFISH probe, in thehybridization buffer, was first directly recorded in situon the microscope and used to optimally set the detec-tion range around the symmetric narrow-shape emis-sion typical of each QD probe. Images were acquiredwith a 63X oil-immersion objective (HCX PL APO, NA1.40) and analyzed with the Leica software. All QDswere excited with a single 405-nm diode laser, and mul-ticolor images were acquired in a simultaneous scan.Unless otherwise specified, the QDs fluorescence wasdetected in a 20-nm spectral window around theiremission peaks. Scanning was performed at a line fre-quency of 200 Hz, averaged two to four times, and theimage format was 512 512 or 256 256 pixels.Autofluorescence of the nuclei was collected through aband pass filter centered at 460 nm to assess nuclear andcellular morphology. Autofluorescence was usedinstead of the more conventional DAPI counterstainingbecause it interfered with QD fluorescence on 405 nmexcitation. Images were further processed with AdobePhotoshop 5.0 (San Jose, CA).

RESULTS

For our studies, we prepared QDDNA FISH probesby incubating streptavidin-coated QDs (Quantum DotCorp.) with single-stranded biotinylated DNA oligonu-cleotides. Formation of the QDDNA complexes wastitrated by electromobility shift assay on agarose gelsand analyzed by multicolor fluorescence analysis usinga combination of 532-nm green laser excitation and640 BP red emission filter to detect the red QDs (605-nmemission) and a 488-nm blue laser excitation and a530 BP green emission filters to detect the single-stranded DNA after counter-staining with SYBR GreenII gel stain (Molecular Probes) (Fig. 1). The conjugationof the DNA on the nanoparticles was demonstrated byfaster mobility of the complexes (caused by a negativenet charge increase) and by co-localization of both theQDs and the DNA fluorescent signals (Fig. 1A). Theslight smearing of the QDDNA complexes onto theagarose gel is likely to be the consequence of variationsin the number of biotin sites (i.e., the number of strepta-vidin molecules) available on each QD that gives rise toparticles with slightly different net charges after coatingof the nanocrystals with the DNA. The size heterogene-ity of the QDs themselves does not contribute to thatphenomenon because bare QDs (without DNA) migratehomogeneously as a single band in the agarose gel (Fig.1A, left lane; Fig. 1B). Moreover, no changes in gelmobility or color co-localization were observed whenthe same but unbiotinylated DNA sequence was used,

which demonstrate that there is little nonspecific DNAadsorption onto the QDs (Fig. 1B).

We then asked if this geometry was optimal to pre-serve the availability of the DNA for hybridization tocomplementary sequences in solution by testing vari-ous combinations of sense and antisense oligonucleo-tide sequences. Only self-complementary sequences(i.e., a sense [S] with an antisense [] sequence) shouldform hybridization aggregates as depicted in Fig. 1C.Indeed, Fig. 1D shows that two colors of QDDNAprobes hybridize to each other only when theirsequences are complementary (lanes 6 and 8, S/ and/S) but not when they are identical (lanes 7 and 9, S/Sand /). The large hybridization aggregates thatresult run as a smear in the agarose gel owing to theirgreat size heterogeneity.

All together, these results demonstrate that QD-DNAprobes are fully functional and solely hybridize to self-complementary targets.

We first tested QDDNA FISH probes against themajor () family of mouse satellite DNA. -Satellites arehigh-copy DNA repeats that localize to centromeric het-erochromatin domains in mouse metaphase chromo-somes (6). Figure 2A,B shows typical images of suchdomains visualized with a -satellite Texas Red probeon an interphase nucleus and a metaphasechromo-some spread, respectively. Large fluorescent foci tend tolocalize at the periphery of the interphase nucleus,whereas the same probe labels specifically every cen-tromere in metaphase chromosomes.

Figure 2C,D shows that the same probe conjugated toQD655 generates qualitatively comparable hybridiza-tion patterns. The QDFISH probe shows good signal-to-noise ratio and comparable penetration/efficiency toits Texas Red counterpart, which ranges from nearly100% targeting efficiency in interphase nuclei to 93.5%on metaphase chromosomes (Table 1). The specificity ofboth the hybridization procedure and the -satellite QD-FISH probe were accessed by performing, in parallel,negative-control hybridizations either with an irrelevantoligonucleotide QD655-FISH probe against the samemouse P815 cell line (Fig. 2E) or by using the mouse -satellite QD655-FISH probe against the human HeLa cellline (Fig. 2F). Changing the oligonucleotide probesequence and the endogenous target sequence both pre-vented proper targeting because no hybridization sig-nals were detected (Figs. 2E,F, respectively). Bothnegative hybridization results demonstrated the speci-ficity of the hybridization of the -satellite QDFISHprobe to its proper mouse target.

To further confirm proper targeting of the -satellitesQD probe, we performed a sequential dual-colorhybridization experiment with two oligonucleotideslabeled with QD592 or with Texas Red. As shown in



Figs. 3C and 3F, co-localization of the two color probesresults in a yellow fluorescent signal for the mergedimages. We found that the majority of the chromosomeswere doubly labeled in any metaphase examined.

Taken together, these experiments demonstrate thatoligonucleotide-based QDFISH probes are able to pen-

etrate intact interphase nuclei or metaphase chromo-somes and detect subchromosomal genetic sequenceswith good specificity. They can also be used simultane-ously with standard dyes.

We have successfully tested a number of other QD col-ors (565, 585, 592, 605, and 611 nm) that performed



Fig. 2. Visualization of centromeric heterochromatin domains by QDFISH. (A) A Texas Red -satellite repeat probe pro-duces large fluorescent foci at the periphery of the interphase nucleus. (B) The same probe labels almost every centromerein metaphase chromosomes. DAPI counterstaining is shown in blue. (C,D) Labeling signals of a -satellite QD655 probe (405nm Excitation, 655 10 nm Emission) merged with autofluorescence (pseudo-colored in blue; 405 nm Excitation, 460 nm 30 nm Emission). (E,F) No -satellite hybridization signals are detected either when the same mouse P815 cells are hybridizedwith an irrelevant QD655-FISH probe (E) or when the human HeLa cells are hybridized with the -satellite QD655 probe (F).Scale bar: 4 m.

equally well in that assay, with the notable exception ofQD525, which showed an irreversible spectral shift onhybridization that precluded its subsequent use in FISH(data not shown). We observed that the fluorescenceintensity of the QDFISH probes markedly decreases dur-ing the hybridization procedure but could be recoveredon re-exposure to bright light. Figure 4 shows a typicalphotoactivation time course of QD655 fluorescence fol-lowing -satellite FISH. On continuous exposure to a 405-nm violet diode laser delivering 10 nW at the sample (asmeasured with a Newport model 1830-C optical powermeter), the fluorescence of the targeted QDs increasedover several intervals before reaching a plateau afterabout 3 min (Fig. 4AE). The fluorescence increase is

extensive and not the result of changes in the intensity ofthe exciting light in the different pictures that were takenunder the same illumination. An average photo-brighten-ing time constant of about 66 s was obtained by fitting theobserved intensity increase of the different QD images oftwo heterochromatin domains (Fig. 4E,F, areas 2 and 3)with a model function converging exponentially to itsfinal intensity asymptotic value after approx 200 s.

These results show that although the fluorescence ofthe QDs apparently dimmed after FISH, they aremarkedly photoactivable, increasing by approximatelytwofold within minutes after exposure to bright light.

Intermittence in emission (known as blinking) hasbeen universally observed for single QDs (7,8) and was



Table 1Summary of Hybridizations of Centromeric Heterochromatin Domains

in P815 Mast Cells

-Satellite hybridization signals

Type of probe Interphase nuclei (%) Metaphase chromosomes (%)

Texas Red labeled 39/39 (100) 187/200 (93.5)QD655 labeled 49/49 (100) 196/210 (93.5)

Fig. 3. Dual hybridization with two interspersed -satellite probes labeled with QDs and an organic fluorophore. (A,D)Texas Red: 543 nm Excitation, 655 20 nm Emission; (B,E) QD592: 405 nm Excitation, 589 5 nm Emission (C,F) Merge withautofluorescence (pseudo-colored in blue): 405 nm Excitation, 460 nm 30 nm Emission Image acquisition of double-stained samples was recorded in sequential order to minimize crosstalk between the different channels. Scale bar: 4 m.

also readily visible within bright regions of hybridiza-tion at the centromere and in interphase nuclei inQDFISH, whereas no such flickering was noted in con-trol slides detected with Texas Red. Figure 5 showsintensity time traces recorded with a CCD camera at theperiphery of centromeric heterochromatin domains thatare typical of CdSe single-molecule QDs (red curves).No blinking was observed outside the hybridizationsignal areas (black curves). We further investigated con-ditions that had been described in the literature to helpsuppress QD blinking by using thiol-containing bufferssuch as 2-mercaptoethanol (ME) (9). Figure 5B showsthat blinking was slightly reduced but not totally sup-pressed in the presence of 140 mM ME.

Having successfully detected one subnuclear targetwith one QDFISH probe, we then asked if we couldsimultaneously detect multiple targets with distinctQDFISH probes. To test this, we further investigatedthe fine structure of the centromere with two-color QDprobes: a -satellite QD592 probe and a minor satelliteQD655 probe that targets another but 10 to 20 times lessabundant class of repetitive DNA sequences also associ-ated within or close to the centromeres (10). The two-color probe hybridization mixture was first spectrallyrecorded directly on the confocal microscope on excita-tion with the 405-nm blue diode laser to characterize(and correct for) any spectral shifts in the hybridizationbuffer. The detection range was set to 10 nm around



Fig. 4. Photoactivation of QD fluorescence following FISH. P815 cells were hybridized with a -satellite QD655 probe tovisualize the centromeric heterochromatin domains. Cells were exposed to 10 nW of 405-nm laser light. Cells were imagedon the confocal microscope and scanned at a line frequency of 800 Hz with no averaging using a 655 10 nm detection win-dow. Time-lapse images were taken every second for 10 min. Selected images are shown at 0 s (A), 50 s (B), 100 s (C), 150 s(E), and 200 s (E). The white dotted circle indicates the border of the cell nucleus. (F) The integrated pixel intensities of twocentromeric heterochromatin domains from the cell nucleus (areas 2 and 3) were plotted together with the total pixel inten-sity of a neighboring control area of equal size (area 1). Areas 2 and 3 point to nuclear QD patches that brighten after illumi-nation, whereas the control area 1 shows no brightening but photobleaching of the background fluorescence. Thephotobrightening time constants were obtained by fitting the observed intensity increase of the time-lapse images with amodel function converging exponentially to its asymptotic value (area 3: t1 = 70.7 9.7 s; area 2: t1 = 62.4 4.6 s). The back-ground or autofluorescence bleaching time constants (t1 = 36.6 s and approx 25 min, amplitude ratio 5:1) were similarlyobtained by fitting the observed intensity decay of a background spot with a sum of two exponential decays. Scale bar: 4 m.

the maximum emission pick for each QDDNA probe.Figures 6B and 6H show color images where both satel-lite-QD592 and -QD655 probes are readily distin-guished in a single exposure after single-wavelengthexcitation at 405 nm. The two satellite probes are inclose proximity, but are spatially independent in theinterphase cell as co-localization appears randomly(Fig. 6B). They do, however, co-localize precisely at the

centromeres of metaphase chromosomes (Fig. 6H). Theminor satellite repeats appear as sharp doublet signalsat the centromeres with the exception of one pair oflarge foci of more intense staining (Figs. 6G and 6H,double and single arrow, respectively) also seen withthe Texas Red control (Fig. 6D,E). At the level of resolu-tion afforded, both satellite repeats appear to bearranged as discrete blocks with little interspersion of



Fig. 5. QD blinking on labeled centromeric heterochromatin domains in P815 cell nuclei. (A) -satellite hybridization sig-nals were imaged with an inverted wide-field Zeiss Axiovert 100 TV microscope equipped with a PL APO 63x/1.4 apertureoil objective (Carl Zeiss MicroImaging Inc, Thornwood, NY) and a CCD camera (Photometrics Coolsnap HQ, RoperScientific, Trenton, NJ). QD655 were excited using a 100 W mercury arc lamp and imaged with a combination of 530-nmlong pass (LP) Excitation, 507-nm dichroic LP (DRLP), and 600-nm LP Emission filters (upper panel). The white dotted cir-cle indicates the border of the cell nucleus. Images were acquired every 100 ms for 100 s in Metamorph 6.3 (MolecularDevices Corporation, Sunnyvale, CA) and intensity time traces were generated using Asterix software (Dr. Xavier Michalet,UCLA) written in LabVIEW 7.1 (National Instrument Corp., Austin, TX). The intersection of the two red lines (corre-sponding to an area of 5 5 pixels) points to the location for which the intensity time trace is shown (red curve, lowerpanel). It is plotted against the background fluorescence intensity measured in a dark area (5 5 pixels) of the nucleus (blackcurve, lower panel). (B) Same experiment following the addition of 140 mM 2-mercaptoethanol (ME). Scale bar, 4 m.


Fig. 6. Double target detection using two colors QD probes and a single wavelength excitation at 405 nm. (A,CE) Dualcolor hybridization with - and minor satellite probes (FITC and Texas Red, respectively). Minor satellites staining appearas a sharp doublet signal at the centromeres (double arrow) with the exception of one pair of large foci of more intense stain-ing (single arrow). DAPI counterstaining is shown in blue. (B,FH) Same experiment with a mixture of QD592 and QD655probe - and minor satellites, respectively. Detection windows used: QD592 (592 10 nm); QD655 (655 10 nm); autofluo-rescence (460 nm 30 nm). Multicolor images were acquired simultaneously. Scale bar: 4 m.

the two satellite DNA sequences with the red minorsatellite signal being located closer to the short arm ofthe acrocentric mouse chromosome than the major satel-lite green signal (Fig. 6FH). Thus, a complex mixture ofQDFISH probes successfully resolved the structuraldomain organization of condensed pericentromericchromatin regions with good spatial resolution despitetheir larger size compared with conventional organicdyes FISH probes. These experiments demonstrate thatQDDNA conjugates allow direct and simultaneousmulticolor detection of subnuclear genetic targets onsingle wavelength excitation.

DISCUSSION

The present study reports a novel rapid and sensitiveFISH method using site-specific DNA sequencesdirectly conjugated to semiconductor QDs. We haveused high and low repetitive satellite DNA probesinvolving high-resolution single and bicolor FISH. Wehave tested different types of cytological preparations,including metaphase and interphase lymphocytes withpositive results.

The performance characteristics of QDs, which aresignificantly brighter and more photostable than thoseof analogous fluorescein and Texas Red dyes (3),allowed us to visualize the process of hybridizationunder a coverslip during the renaturation of the probeand target DNA in situ and therefore to control both theefficiency of hybridization before the termination of theexperiment and the efficiency of the washing of slides bymicroscopic inspections; and analyze the specimensunder the microscope for minutes to hours. The use ofdirectly labeled QDDNA probes offer several addi-tional advantages for FISH studies because of the elimi-nation of secondary detection steps and the visualizationof hybridization results in a one-step procedure; thelow background fluorescence; and the increased poten-tial for multicolor probe detection. Indeed, our directlylabeled QDDNA probes approach provides the firstexploration of QDFISH multiplexing applicability. Theonly other published study that detects the hybridiza-tion sites in situ of biotinylated probes with QDs usedthem as a secondary reagent and thus is intrinsicallylimited to single-color detection (3) because it implies,as in conventional FISH, that multiple DNA probesmust be detected in separate, nonoverlapping detectionschemes to prevent their cross-detection. The same lig-andreceptor binding pair (i.e., biotinylated probes andstreptavidin-functionalized QDs) cannot easily allowthe detection of multiple probes with multiple QD col-ors. Each new color in the detection scheme wouldrequire QDs functionalization with a new hapten. Onthe contrary, our approach, which relies on preforma-

tion of the QDDNA probes (by direct attachment of theDNA onto the QDs), allows the same biotinstreptavidininteraction to be used to generate an infinite array ofDNA FISH probes that can be used in a single step todetect multiple subnuclear genetic sequences with mul-tiple colors. This strategy replaces the use of multiplehaptens by one universal binding pair.

Despite being 10 to 20 times larger than dye-labeledprobes, QDDNA probes performed well in targetingdense and highly compact centromeric heterochromatindomains. FISH efficiency is high because virtually mostinterphase nuclei and metaphase chromosomes harborsatellite FISH signals (see Table 1). This is in sharp con-trast with a previous study by Xiao et al. showing thatmost centromeric regions on human metaphase chro-mosomes could not be detected with QD labels (3). Wewould like to suggest that this unexpected difference insensitivity/efficiency of signal distribution is likelycaused by variable steric hindrance constraints operat-ing at different time along the FISH procedure. Indeed,whereas heterochromatin-embedded probes might besheltered from the QD labels during post-hybridizationdetection because of steric hindrance (3), QDDNAprobes have greater access to the unwinded double-stranded DNA target at the time of hybridization (i.e.,this work). Taken together, our results support thenotion that steric hindrances have little effect at the timeof annealing of the QDDNA probes to its target butmay limit QDs access at the time of detection.

Semiquantitative analysis of fluorescent imagesusing a CCD camera showed that the number of QDspresent at the hybridization sites varied from approx 10to 250 QDs (Table 2 and data not shown) depending onthe size polymorphism of the centromeric repeat arraysof the different mouse chromosomes. Despite the factthat the absolute length of these long, uninterruptedtandem arrays is not known (because of an inherentmapping/assembly problem after sequencing), thenumber of targeted QDs appears far less than the esti-mated several thousands of tandem repeats of satelliteDNA monomers organized in discrete blocks at the cen-tromere of mouse chromosomes (6,10). Three main fac-tors might account for the apparent paucity of QDs seenat the FISH signals. First, it is possible that the bulkynature of the QDs prevent them from homogenouslytargeting the entire three-dimensional structure of thesatellite array despite their overall great efficiency (seeTable 1). Second, fluorescence correlation spectroscopyanalysis in solution has revealed that up to 50% of allQDs are in a dark state and do not fluoresce (11). Last,the recorded total signal intensity can be affected by thestochastic QD blinking behavior within FISH signalsand the power law distribution of on and off time thatis characteristic of QDs (12). This means that long inte-gration time will not increase the brightness of a given



spot linearly with time, precluding any reliable probequantification based solely on intensity measurements.In other words, two different FISH signals resultingfrom approximately the same number of QDs may stillshow variability in brightness in ensemble imaging.

Addition of 140 mM ME did not overcome thatintrinsic limitation of QD blinking similarly to what hasbeen also recently observed in fixed, permeabilized cellsthat had endocytosed QDs (13). Yet, Hohng and Ha haveshown that blinking suppression was already efficient at1 mM ME on bare, spin-coated QDs and suggested thatthe reduction of blinking was a direct result of the thiolgroups binding to the ZnS surface (9). This discrepancymight thus indicate some accessibility issues of smallthiol molecules in fixed cytological materials.

In the course of these experiments, we observed apartial loss of fluorescence of the QDFISH probes.However, bright fluorescence intensity can be fullyrecovered on re-exposure to UV light, similar to whathas been observed for QDs in nonaqueous solvents (14)and, more recently, inside fixed mammalian cells afterQD endocytosis (13). As expected, the photo-brighteningtime constant (approx 66 s) appears to be an intrinsicproperty of the QDs, independent of the size of the tar-geted heterochromatin domains and thus of the numberof QDs at the hybridization site. By contrast, the fluores-cence saturation intensity value is directly proportionalto the size of the heterochromatin regions and reflectsthe number of QDs targeted in situ (Fig. 4F).

The ultimate resolution in cytogenetics is reached bywiping out nuclear organization altogether and con-ducting FISH on DNA fibers that have been affixed toglass (1518). Although morphological information is

lost, what was condensed in an interphase or metaphaseFISH spot becomes a long fluorescent line in fiber-FISH.Because QD blinking demonstrates single QD detectionbound to chromosomes, it should then be possible toapply QDFISH probes in fiber-FISH to analyze the stillpoorly understood structural organization of long tan-dem repeat arrays or to detect small-scale rearrange-ments in chromosomes. Given that the same laserwavelength excites all QD probes, the effects of chro-matic aberrations are minimized. Thus, in principle, theconcomitant development of ultrasensitive microscopeinstrumentations will allow the use of QDFISH probesfor ultra-high-resolution co-localization of multicolorfluorescent probes with nanometer accuracy down tothe single DNA molecule (19) or single QD detectionlevel (20).

In summary, we have demonstrated simultaneousmulticolor FISH imaging down to the subchromosomallevel using QD-based probes. Coupling hybridizationwith detection without the need for laborious sec-ondary amplification results in a faster, single-step FISHprotocol. The biotin-streptavidin interaction can beused to generate an infinite array of QDDNA FISHprobes and the possibility of using them in a commonhybridization mixture. Despite their larger size, QDprobes show good targeting of dense chromatin struc-tures and work well in conjunction with organic dyesunder standard experimental FISH conditions. Theresults of this study indicate that QDFISH probes maybe applied as additional tools in molecular cytogeneticsand may offer significant benefits in medical diagnostic.QDFISH probes could be used in basic and clinicalcytogenetic studies, both of which require high-resolu-tion and multicolor FISH. Indeed, this new FISHmethod opens up new opportunities for the develop-ment of true color distinctive banding patterns for eachchromosome and shows the proof-of-principle of multi-plex FISH with spectrally distinguished QDs, ratherthan complex probe mixtures, which require sophisti-cated digital imaging analysis and pseudo-coloring(2123). Further work with these new types of FISHprobes will be needed to extend the potential of QDs toRNA detection of specific mRNA transcripts as well.

NOTE ADDED IN PROOF

As this manuscript went to press, Chan et al. pub-lished a similar QDFISH method for the multiplexdetection of mRNAs (24). Recent results by Xiao et al.suggest that pH effects on fluorescence of QD-detectedhybridization signals could account for the absence ofQD centromeric hybridization signals on humanmetaphase chromosomes in their FISH experiments (25).



Table 2 Estimated Number of QDs Hybridized onCentromeric Heterochromatin Domains in P815 Mast

Cells

-Satellite Hybridization Signalsb

Nucleia 1 2 3 4 5 6 7

Cell 1 9c 39 42 14 15 26 49Cell 2 57 48 NA NA NA NA NA

a Same as those displayed in Fig. 5.b Numbering starts at the intersection of the red cross in

Fig. 5 and goes clockwise.c The estimated number of QDs was calculated by dividing

the total intensity counts of a given centromeric heterochro-matin domain by the averaged intensity count of a single QD(assumed here to be constant) extracted from the intensitytime traces minus the total pixel intensity of a neighboringcontrol area of equal size.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Xavier Michalet,for his critical reading of this manuscript as well as helpwith data analysis, and Tal Paley for editorial assistance.Fluorescent microscopy was performed at the CNSIAdvanced Light Microscopy/Spectroscopy SharedFacility at UCLA. This work was funded by the NationalInstitute of Health, grant no. R01 EB000312-04.

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