are used, either to block transcription of genes within thenucleus of cells or to block translation of messenger RNAwithin the cytoplasm (1,2). Accordingly, if radiolabeled,these oligonucleotides may usefully carry radioactivity totargeted cells or tissues. This and other applications ofthese reagents as radiopharmaceuticals, however, will require that methods be developed for radiolabeling themwith diagnostic and, possibly, therapeutic radionuclides.Methods for radiolabeling oligonucleotides with beta-emitting radionuclides such as 3H,@ and 32P are well established (3—6);however, the use of oligonucleotides radiolabeled with imageable radionuclides is in its infancy.Methods have been reported for labeling oligonucleotideswith ‘@I(6,7) and, as such, these methods should be usefulwith ‘@Iand ‘@‘Iradionuclides with imaging properties.Depending on the pharmacokinetic properties of an oligonucleotide of interest, it is likely that the imaging radionuclide of choice will often be 99mTc Recently, a method forradiolabeling DNA with @Tcwas described which reliesupon a derivative of DTPA attached to the oligonucleotideto form chelates with reduced @‘@‘Tc(8). DTPA has previously been used for radiolabeling antibodies with @“Tcbutwas abandoned when the instability of the label was judgedto be unacceptably high (9). Accordingly, this study wasperformed to investigate an alternative approach to labeloligonucleotides stably with @Tc.A hydrazino nicotinamide (SHNH) moiety has been shown to form stablecomplexes with @Tcwhen conjugated to antibodies (10).In this investigation, an amine-derivatized DNA was conjugated with SHNH and the properties of the label evaluated. To provide a useful comparison, the same DNA wasalsoconjugatedwithDTPA,as hasbeendescribedbyothers (2,11), for radiolabeling with “Inusing proceduresroutine for antibody labeling (12).

In this investigation, 22-base, single-stranded DNASwereselected because their small size (about 8 kDa) was expected to facilitate rapid whole-body clearance after administration. Moreover, a 22-base oligonucleotide is largeenough to virtually exclude any possibility of an accidentalmatch within the genomic DNA and its transcripts (1).DNAS used were derivatized on one end with a biotinmoiety to provide a useful means of establishing labelingefficiency and label stability. Using this feature, radiolabeled DNAS may be easily distinguished from unbound or

@ngIe-strandedRNA and DNA oligonucleotides may be usefulas radiopharmaceuticais for antisense and other in vivoapplications ifconvenient methods for stably attaching radionuclidessuch as @‘Tccan be developed. Methods: To radiolabel DNAwith @‘1c,we have used the hydrazino nicotinamkle (SHNH)rnolety devebped elsewhere. The diethylenetriaminepentaacetic acid (DWA) chelate was used to label DNAwith 111lnforcomparison. Complementary 22-base, single-stranded oligonucleotides were obtained, each wfth a pnmary amine attached toeither the 3' or 5' end and witha biotinmoietyon the oppositeend. The DNA was conjugated with SHNH by a N-hydroxysuccinimide derivative and with DTPA by the cyclic anhydride.Results: Reversed-phase HPLCanalysis showed that essentially complete conjugation was achieved in both cases. ThepurifiedSHNH-DNAwas radiolabeledwith @rcby transchelation from glucoheptonate at labeling effiCiencieSof up to 60%and DTPA-DNA @4th111Inacetate at up to 100% efficiency.After labeling, the ability of the DNAs to bind to streptavidinthrough the biotinmoieties and to hybridIzewith their compiementary DNAin saline was retained for both radiolabels asdetermined by size-exclusion HPLC analysis. HPLC radiochromatograms of serum incubates showed a shift of @‘@‘Tc,but not1111n,to a high molecular weight, strongly suggesting serumprotein binding in the fomier case only. Low-molecular wsightdegradation products were seen with ‘11ln,but not with @Tcand may be related to the use of phosphodiester-linked oligonucleotides. As a further measure of label stability, the DNAswere bound to strepta@Adin-conjugated magnetic beads andincubated in fresh 37°Chuman serum. Less than 4% of @Fcand 14% of 1111nwas lost in 24 hr. Conclusion: Amino-modifled,single-stranded DNAcan be stably radiolabeledwith @1cby the SHNHmoietywithout loss of function.

Key Words oligonucleotides;technetium-99m;radiolabeling

J NuciMed1995362306-2314

he extraordinary properties of DNA and RNA suggestthat there is potential for the use of these oligonucleotidesas radiopharmaceuticals. For example, there is current interest in antisense applications in which oligonucleotides

ReceivedAug.4,1994;revisionacceptedDec.29,1994.For correspondence or reprints contact: D.J. HnatOWich,PhD, Department of

NudearMedicine,Universftyof MassachUsettsMed@alCent&,Worcester,MA01655.

2306 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995

Technetium-99m Labeling of DNAOligonucleotidesD.J. Hnatowich, P. Winnard Jr., F. Virzi, M. Fogarasi, T. Sano, C.L. Smith, CR. Cantor and M. Rusckowski

Department of Nuclear Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts, and Center for AdvancedBiotechnology and Departments of Biomedical Engineering Phartnacolo@jiand Biology, Boston University, Boston, Massachusetts

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0

@ 0 00 BrnN@ oxo,_@

FiGURE1. Structureof A and B DNAchains used in this investigation.

dissociated label by size-exclusion analysis before and afterthe addition of streptavidin (13).

In this study, we describe the results of conjugating 5everal single-stranded DNAS with SHNH and DTPA followed by labeling with @Tcand ‘‘‘In,respectively. Thestability of the label was determined in serum, and biodistribution studies were performed in normal mice.

MATERIALS AND METhODS

Four 22-base, single-stranded DNA sequences were purchased(Operon Technologies,Alameda,CA) for this investigation.Thebase sequences were 5'-biotinTA ATA CGA CFC ACF ATAGGG AGamine-3' (A-chain) and 5'-amineGG TAC AGG TCfCAC TGT ATG ACbiotin(C-chain)and their complements(B-and D-chains, respectively). Figure 1 presents the structure of theA- or B-chain (i.e., with the biotin moiety on the 5' end). Asshown, the biotin moiety was attached directly through a 15-member amide-polyether linker to the terminal phosphate, whilethe amine was normally attached to the terminal phosphate groupthrough a 6-member methylene carbon spacer. The molecularweight of each chain was about 8.2 kDa. The melting and annealing temperatures in physiologicalsaline were calculated to be57—60°Cand 32—41°C,respectively, for both pairs (14). The DNASwere purchased unpurified and were used without further purification. They were generally handled under sterile conditions; allsolutions were sterilized by terminal filtration through a 0.22-smfilter, and sterile pipette tips were used. All other pipette tips andtubes were autoclaved prior to use.

Streptavidin was purchased and used without further purification. The DNAs were stored dry as received, at refrigerator tern

peratures and were dissolved at a concentration of 1—4mg/ml insterile water when needed. After solubilization, 20—500p.g DNAwere added to sterile plasticvialswhichwere immediatelyfrozenfor storage. Technetium-99m-pertechnetate was obtained from a

@Mo@@mTcradionuclide generator and ‘‘‘Inwas purchased as thechloride complex. Streptavidin-conjugated superparamagneticpolystyrene beads (Dynabeads M-280, Dynal, A.S., Lake Success,NY) were stored at refrigerator temperatures as recommended bythe manufacturer. The N-hydroxysuccinimide (NHS) derivative ofthe hydrazino nicotinamide moiety was a gift from Dr. M. Abrams,

(JohnsonMattheyInc., West Chester,PA). The cyclicanhydrideof DTPA was synthesized as previously described (15).

Oligonucleotide ConjugationIn this investigation,all four DNAchainswerederivatizedwith

either SHNH or DTPA. In the former case, the NHS-derivative ofSHNH moiety was dissolved in dry dimethylformamide to a concentration of 8.6 mg/ml (10). For conjugation, the DNA solutionwas diluted with a sterile bicarbonate buffer so that the finalconcentrations were 2.0 mg/ml DNA, 1M NaCl, 0.25 M NaHCO3,1.0 mM EDTA at pH 8.3—9.0.The EDTA was added to complexcations such as calcium. The DNA solutions were incubated for 30mmat 45°Cjustprior to SHNHadditionto dissociateanysecondary complexes between the primary amines and the phosphatebackbone.Between0.2 to 1mgof DNAwasconjugatedat a 1—25molarexcessof SHNHto DNAbyaddingthe necessaryvolumeofdimethylformarnidesolutiondropwiseto the DNA solutionwhilevortexing.The solutionwas incubatedat room temperature for 1hr. Followingincubation,the conjugatedoligonucleotidewaspurifled on a 0.7 x 20 cm gel filtration column of P-4 using a sterile0.25 M ammonium acetate, 1.0 mM DTPA buffer at pH 5.2 aseluant. The DTPA was added to complex excess stannous ion andhelp prevent radiocolloid formation. Fractions (0.4 ml) were collected and the absorbance (260 nm) of each measured. Oligonucleotideconcentrationswere estimatedusingan extinctioncoefficient determined in this laboratory of 30 @.d/@gfor a 0.1% solutionmeasuredat 260nm.The absorbanceof SHNHat thiswavelengthand under these conditions was found to be insignificant. Thefractions collected contained DNA at concentrations in the 0.5—1.0mg/mirange.

ConjugationwithDTPAwasachievedwiththe cyclicanhydrideas previously described for proteins (12). Approximately 1 mg ofthe oligonucleotide was dissolved in 10 ml 0.4 M HEPES buffer,pH 8.5, and this solution was added rapidly to a round-bottom testtube containing 100 mg of the thy anhydride while vortexing. Thus,the DTPA:DNAmolar ratio was 2110:1.The solutionwas incubated at room temperature for 30 mm. The conjugatedoligonucleotides were originally purified on a gel-filtration column of P-2using 10% ethanol as an eluant, but the inability to remove anunidentifiedradiochemicalcontaminantusingthis columnnecessitatedthe useof an alternativepurificationmethod.The reaction

Labeling DNA Oligonucleotides with Technetium-99m •Hnatov@chat al. 2307

T c@,o 3.ri,N@L0X0@ 120

0 0 B.s•N

0'

NH,

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mixture was adjusted to pH 3.5 using glacial acetic acid and waspurified on a 1.7 x 9-cm column of DEAE Sephadex in the acetateform (Sigma) by stepped elution. The first 50 ml of eluantwere 1%acetic acid. The next 90 ml of eluant were 1% ammonium acetate,pH 5.5,inwhichthe concentrationof NaClwasincreasedfrom0.0to 0.5 M in five equally spaced steps. The final eluant consisted of20 ml 1% acetic acid, 0.5 M NaCl. The oligonucleotideswereeluted between 139—153ml as determined by absorbance at 260nm and by polyacrylamidegel electrophoresis.The peak fractionswere pooled, aliquoted into small fractions and stored frozen.

The extent of both SHNH and DTPA conjugation to oligonucleotides was determined by reversed-phase high performanceliquid chromatography (HPLC) using a 4 X 250-mm C-18 column(BioRad), a flow rate of 0.8 mI/mm and a linear gradient over 40mm from 0.07 M NaClO4 to 100% acetonitrile. Unconjugatedoligonucleotides elute with a retention time of about 12 mm, whilethe SHNH-conjugated oligonucleotide elutes at about 21 mm.Free DTPAelutesat or near the voidwhilethe DTPA-conjugatedoligonucleotide elutes at about 3 mm.

OI@onudeobde LabelingThe SHNH-conjugatedoligonucleotideswere radiolabeledby

transchelation from @Tc-glucoheptonateas has been previouslydescribed for the labeling of mercaptoethanol-reduced antibodieswith @“Tc(16). Several vials were prepared under nitrogen, eachcontaining200mgglucoheptonicacidand 140 @gSnCI2-2H20 ata final pH of 5.5 and were lyophylized. For reconstitution, 3 mlnitrogen-purgedsalinewasaddedthroughthevialseptum.Prior toradiolabeling,the SHNH-conjugatedoligonucleotidesolutionafter P-4 purification was incubated for 10—15mm at 45°Ctodissociate any secondary complexes. Between 5—10 @Ci[ @Tc}penechnetateeluantwasaddedtoeachmicrogramofconjugated oligonucleotide. Immediately thereafter, 20 @dof the reconstituted glucoheptonate kit was added (i.e., 1.3mg of glucoheptonic acid and 1.0 p.gof SnCl2 2H20) for @“Tcactivities up to 1mCi. The solution was incubated for 1 hr at 45°C.The labeledoligonucleotide was then purified on a 0.7 x 20-cm gel filtrationcolumnof Sephadex0-50 usingsterilesalineas eluant. Radioactivity as well as absorbance at 260 nm was used to identify andquantitate peak fractions. As a control, the identical labelingprocedurewasappliedto the unconjugatedoligonucleotide.

Late in this investigation, glucoheptonate was replaced withtricine as transchelator (17). A fresh sterile solution of tricine(Sigma) in 0.02 M NaOH, pH 7.0, at a concentration of 70 mg/mlwaspreparedandmixedwitha freshsolutionofstannousionin0.1M HCIso that 1.4 @dof the tin solutionwasaddedto each 150 @dof the tricine solution. Typically, an aliquot of this solution wouldbe added to a solution of pertechnetate and SHNH-conjugatedDNAso that the finalconcentrationswere 18mCi/ml[@“TcJpertechenetate, 0.05 mM DNA, 0.14 M tricine, 0.15 mMSnCl2-2H20, pH 7. The labelingsolutionwas incubatedat roomtemperature for 10mm before purificationby 0-50.

The DTPA-conjugatedDNA was radiolabeled after DEAFpurification with ‘‘‘In-labeledacetate, as is routine for labelingDTPA-coupledantibodieswiththisradionuclide(12).The acetatecomplexwaspreparedbyadding1M sodiumacetate,pH 6, to theacidic ‘111n-chloridesolution. Up to 180 p@Ci @‘Inwere added to1.6 @gDNA.The labeledDNAwaspurifiedon a 0.6 X 20-cmgelfiltration column of Sephadex 0-25 using sterile saline as eluant.Radioactivityaswellas absorbanceat 260nmwasused to identifyand quantitate peak fractions. As a control, the identical labelingprocedure was applied to the unconjugated oligonucleotide.

A denaturing20%polyacrylamidegelwasusedfor the analysisof DNAwitha 89 mMIris borate, 2 mMEDTAbufferat pH 8.3(ThE) (18). Samplesfor analysiswere preparedin ThE containing 40% formamideand 0.05% each of bromophenolblue andxylene cyanole. The solution was heated at 80°Cfor 10 mm priorto loading on the gel. Each lane contained 100—400ng DNAadded in 1—5@dand the electrophoresiswasperformedat 12mAunderconstantwatercooling.The separationwascompletedwhenthe bromophenol blue reached within 1 cm of the gel bottom.After removal from the apparatus, the gel was stained with 0.5

@g/mlaqueous solution of ethidium bromide for 10 mm and thenwashed in water for an additional 10 mm. The gel was then placedin a UV lightboxand photographedwith Polaroid665film.

Preparation of Streptavidin-DNA ConstructsSize-exclusionHPLC analysisbefore and after the additionof

an unlabeled construct consistingof the complementaryDNAchains bound by their biotin groups to streptavidin was also usedto demonstratethat the label wasbound to DNA.The extent ofthe shift in the radioactivityprofile to a higher molecularweightafter the addition is a measure of radiochemicalpurity. Thestreptavidin-DNAconstructswereprepared byaddingthe desiredDNA chain at a 1:1molar ratio relativeto streptavidinin saline,pH 6, and incubatingfor 30 mm at room temperature.The constructs were used without purification.

Serum incubation Stud@sAfter radiolabeling with @“Tc,C-chain was added to fresh

human serum at a concentrationof 1.2 @.tg/mland incubated at37°C.Samples were periodically removed for size-exclusion HPLCanalysis using a single 1.0 X 30-cm Superose-12 column (Pharmacia) and 0.1 M sodium phosphate buffer with 0.15 M saline (PBS)eluant, pH 7.0. The stability of both labels in serum was determined for the radiolabeled C-chain which had been bound,through its biotin moieties, to streptavidin-conjugatedmagneticbeads. In preparation, suspensions of beads were washed threetimes with a washing buffer consisting of 10 mM Tris, 1 M NaCl,0.5 mM EDTA and 0.05% Tween 20, adjusted to pH 7.5. The

beads were manipulated for washing by using a magnetic separator. After the third wash,the beadswere blockedwith a blockingbuffer consistingof the washingbuffer containing0.1% calf thymus DNA. The calf thymus DNA averaged 550—750nucleotidesand was stored frozen in a 10-mg/ml aqueous solution. It wasdiluted 1:10 for use with the washingbuffer. The beads wereincubatedin the blockingbuffer for 30 mm at room temperaturewith occasional shaking to block nonspecific DNA binding. Theblockingbuffer was removed and the beads resuspendedin thewashingbuffer.The suspensionwasthen dividedequally.One-halfof the samplewasblockedwithbiotinasa controlbyincubatingfor15mm at room temperaturewith a 100-foldexcessof biotin overthat necessaiyfor saturationas statedbythe manufacturer(i.e.,73,.Lg biotin per ml of beads at their original concentration). To each

half of the samplewas then added the labeledconjugatedoligonucleotides at a concentration which was about 50% of that necessary for saturation of the streptavidin as stated by the manufacturer (i.e., 19 @gof a 22-baseoligonucleotideper ml of beads attheir original concentration). The beads were incubated with agitation for 30 mm at room temperature and were then washed fivetimes with the washing buffer. The washing buffer was then removed and fresh human serum added to the “dry―beads. Thebeads were resuspendedand placed in a 37°Cshakingplatform.Aliquots were removed periodically; the beads were separated and

2308 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995

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IC)c\2

0z

0Cl)

0.6

@ 0.5

RESULTS

Oligonucleotide ConjugationIn this study, the amine-derivatized DNAS were conju

gated with an NHS derivative of SHNH and with the cyclicanhydride of DTPA. Figure 2 presents UV absorption chromatograms obtained by reversed-phase HPLC of unmodifled A-chain (panel 1), SHNH-conjugated A-chain prior topurification (panel 2), and DTPA-conjugated A-chain afterpurification (panel 3). The unmodified A-chain is clearlyresolved into one major peak at a retention time of about12 mm and several minor peaks.

Conjugation with SHNH was accomplished at molar ratios of SHNFI:DNA of between 1—25:1.No differences inthe ability of the conjugated oligonucleotide to hybridize orto accept a @Tclabel were observed at these molar ratios(data not presented). Accordingly, all subsequent conjugations were performed at a 1:1 molar ratio.

After conjugation with either SHNH or DTPA, the presence of unmodified A-chain was reduced to a minor constituent (Fig. 2). The peak absorbance shifted to 21 mm inthe SHNH case and 3 mm in the DTPA case. The unmodified DNA peak is essentially absent, demonstrating thatconjugation was largely complete.

Because DTPA-coupled oligonucleotides purified on aP-2 column contained unacceptably high levels of unidenrifled radiochemical contaminant(s) after radiolabeling; purification was ultimately achieved on an anion exchangecolumn. Figure 3 shows the UV absorbance profiles offractions off this column. The high acidity (pH 2.7) of theinitial eluant apparently results in complete protonation offree DTPA which then elutes from the column early. TheDNA, however, still possesses a high negative chargethrough its phosphate groups and is retained. As the ionicstrength is increased, however, the increasing sodium ionconcentration apparently neutralizes this charge and permits the DNA to elute. The three major peaks (eluting at20, 100 and 150 ml in the figure) were analyzed by gel

0 @0 20 30 40

TIME (mm.)

FiGURE2. UV absorptionchromatogramsobtained by reversed-phase HPLC analysis of unmodified A-chain (trace 1), ofSHNH-conjugated A-chaln prior to purification (trace 2) and ofDTPA-conjugated A-chaln prior to puiiflcation(trace 3).

washed three times with the washing buffer. The serum, the combined washes and the beads were then counted in a NaI(Tl) wellcounter.

AnimalBiodistñbutionShiduesBiodistributions of both @“Tc-and ‘DIn-labeledD-chain were

determined in normal CD-i male mice. Each animal received bytail vein administration0.1 ml of saline containingeither 7.5 @g(7.3 MCi)“In-or 10@ (7 MCi)99mTc..labeledDNA. Animalswere killed by spinal dislocation 2.5 hr postadministration. Samples of organs were rinsed in cold saline and were counted alongwith a blood sampleand an aliquot of the injectate in a NaI(Tl)wellcounter.The biodistributionswerereportedas the percentageof administered radioactivity per gram of tissue.

vol.(ml)0—49

50—144

145—160

1% HAc, pH 2.717@NH 4Ac. pH 5.5

1% HAc, pH 2.7

0.5

0.4

z0.3@

0.1

0.0

a0

c,i

FiGUREa uv absorptionprofileof freetions obtained by anion exchange chromatography of DTPA-conjugatedC-chsin. Ofthethree major peaks (at 20, 100 and 150 ml),only the latter was shown to contain DNk

:: r\ @:@@ /1

:@@@ @JJ@@/

0.0 --/@ .@

0 20 40 60 80 100 120 140 160 150

VOLUME (ML)

2309Labeling DNA Oligonucleotides with Technetium-99m •Hnatowich et al.

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FIGURE 4. Radiochromatograms obtalned by size-exclusionHPLCanalysis of99mTc@labeiedC-chain (leftcolumn)and 111lnlabeled C-chain (right column) in saline (trace1), in saline containing biotin-saturatedstreptavidin (trace 2), in saline containingstreptavidin (trace 3) and in saline containingcomplementary D-chain bound to streptavidin (trace 4).

electrophoresis. Only the latter peak contained DNA (seebelow). The recovery of DNA in this purification was 16%.

Oligonucleotide LabelingWhen 100@ or more of the SHNH-conjugated DNAS at

a concentration of at least 250 @tWmIwere radiolabeled withup to 1 mCi @°@Tc,labeling efficiencies ranged between30%—60%,as determined by Sephadex 0-50 chromatography. Attempts to label smaller amounts of DNA or at lowerconcentrations resulted in reduced efficiencies. These observations were independent of the DNA chain. As controls, the unmodified DNAS were radiolabeled in the identical fashion. Labeling efficiencies were less than 5% in thecase of each 99mTc control. When the @“Tc-labeledoligonucleotides were analyzed by size-exclusion HPLC, a singlepeak was always observed. Recoveries occasionally approached 100%, although typical recoveries were 81% ±5% (s.d., n = 4). That recoveries could be increased byincreasing the salinity of the eluant suggests that this retention was due, in part, to ionic interactions of the chargedDNAS with the column support.

Typically, specific activities of 50 @Ci4tgDNA wereachieved when glucoheptonate was replaced with tricine.Properties of the labeled DNAS such as biotin binding,hybridization, protein binding in serum or serum stabilityappeared to be unchanged with respect to DNA labeled viaglucoheptonate.

Labeling efficiencies of the DTPA-conjugated andDEAE-purified oligonucleotides approached 100% undermost conditions of DNA concentration and at specific ac

0U)

tivities of 60 @Ci/.tg.The unconjugated C-chain underidentical conditions retained only 2% of the “Inlabel.Figure 4 presents size-exclusion HPLC radiochromatograms of C-chain radiolabeled with 99mTc(left column) and“In(rightcolumn)before(trace1) andafter(trace3) theaddition of excess streptavidin. Both labeled DNAS elute ina single peak which largely shifts to a higher molecularweight (i.e., smaller elution volumes) with the addition ofthe streptavidin. Similar shifts to high molecular weightwere observed with the addition of the streptavidin-D-chain(complementary) construct (trace 4). Also in Figure 4 areradiochromatograms showing no shift with the addition ofbiotin-saturated streptavidin (trace 2). Recoveries in theseHPLC analyses were always 80%—90%.Because[ @TcJpertechnetateisretained,thegoodrecoveriesdemonstrate that oxidation of the label to pertechnetate was notan important mode of instability.

Elecfro@ ShidiesThe first four lanes in the electrophoretogram of Figure

5 contain unmodified DNA chains A through D, respectively, stained with ethidium bromide. Whereas chains Cand D show only one band at the position expected for a22-base DNA, that of chains A and B show two distinctbands, one co-migrating with chains C and D plus an additional band with an apparently lower molecular weight.Several other minor bands corresponding to higher molecular weight have occasionally been seen as well. Lanes 5—7contain aliquots of three peak fractions (at 20, 100 and 150ml, respectively) from the DEAE anion exchange purifica

99m 111 In

ELUTION VOLUME

2310 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995

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Injectate,.11.0

. IHour

Ir.1.@)L..

@ . . .

1 5 10 99mTC 1111n

00

FIGURE5. PAGE electrophoretogram shows DNA bandsstained withethidium bromide. Lanes 1-4 are unmodifiedA-, B-, C-and D-chains, reSpeCthiely,and show the presence of DNAimpur@rtiesin the A- and B-chain preparations. Lanes 5-7 are aIk@uotsoffractions at 20, 100 and 150 ml, respecThiefy,of an anion-exchangecolumn in the purificationof DTPA-coupledD-chain. Onlythe latterfraction contains DNA.Lanes 7 and 8 are D-chain conjugated withDTPA without (lane 7) and with (lane 8) the addrtiOn of strepta@din.Lanes 9 and 10 are 0-chain conjugated with DTPAwithout (lane 9)and with Qane10) the addition of streptavidin.

tion of DTPA-coupled C-chain (Fig. 3). Only in lane 7 isDNA evident. Accordingly, only the peak eluting at 150 mloff the DEAE column contains DTPA-coupled DNA. Thatthis oligonucleotide has retained its biotin moiety is evidentfrom lane 8 which contained an identical aliquot of thethird DEAF peak but mixed with excess streptavidin. Allthe DNA has shifted to the top of the lane and is thereforeassociated with a high molecular weight species. Similarly,lanes 9 and 10 are of SHNH-conjugated C-chain with andwithout the addition of streptavidin, respectively. As before, a complete shift to higher molecular weight is evidentafter the addition of the protein to the biotinylated DNA.

Serum Incubation StudiesFigure 6 presents size-exclusion HPLC radiochromato

grams of serum incubates of C-chain labeled with @“Tc(left column) and “In(right column) that show the radiochromatographic profiles before (top panels) and after 1 hr(middle panels) and 24 hr incubations (bottom panels) in37°Cfresh human serum. A rapid shift to a higher molecular weight (i.e., smaller elution volumes) is evident onlywith @Tcand almost certainly signifies serum proteinbinding. This binding was seen with each oligonucleotidesequence tested. Several studies were performed to establish whether the label had dissociated from DNA prior tobinding to serum protein(s). The serum samples were made25 mg/mI in calf thymus DNA before the addition of labeled DNA. Repeat size-exclusion HPLC measurementsafter a 1-hr incubation showed an increase of about 40% ofthe radioactivity co-eluting with the labeled oligonucleotides. In additional experiments, an excess of streptavidinconjugated beads was added to the serum and, after a 2 hr

0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 607080

Fraction

FIGURE& Size-exdusionHPLCradiochromatogramsof serumincubates of C-chain labeled with @‘Tc(leftcolumn)and 111ln(tightcolumn)show the radiochromatograph,c profilesbefore (top panels)and after 1 hr (middlepanels) and 24 hr incubation (bottom panels)in 37°Cfresh human serum.

incubation with agitation, the beads were washed andcounted. About 40% of the @Tcwas bound to the beads.Although the binding was not complete, the 40% must becomparedwith a value of about 35%—60%of @“Tc-DNAwhich binds to beads under identical circumstances whenincubated in saline. The 40% binding from serum thereforeshows that the 99mTclabel was bound, at least in part, toone or more serum proteins as the labeled DNA.

Because the serum protein binding of free DNA complicates the determination of serum stability, the oligonucleotides were incubated in serum while bound to streptavidinbeads. When presented to the streptavidin-conjugated magnetic beads as described above, 62% ±1% (s.d., n = 5) of

@“Tcand 51% ±1%(s.d., n = 5) of “Inwas bound.Nonspecific binding, measured with biotin-saturated beads,was 0.7% ±0.4% (s.d., n = 5) for both labels. Figure 7presents the percentage of @Tcand “In bound tostreptavidin-conjugated beads which was released duringserum incubation at 37°C.The figure is based on a singlepreparation of both @“Tc-and “In-labeledC-chain witheach preparation added to five serum samples. Only3.7%±3.7%(s.d.,n = 5) of @‘Tcand13.5%±4.8%(s.d.,n = 5) of “Indissociatedfrom the beads in 24 hr ofincubation.

2311Labeling DNA Oligonucleotides with Technetium-99m •Hnatowich at al.

24 Hour

. ]@@

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111ln@@TcOrgan

% lD/g s.d. % big s.d.

Resultspresented as percent injecteddose per gram (% big) with1s.d.ofthemean.Thelevelsof @1cinalltissuesaresignificanflyhigherthan 111ln(p < 0.001, Student's unpairedt-test).

useful for in vivo imaging applications. Recently, a methodof radiolabeling oligonucleotides has been reported inwhich a derivative of DTPA is covalently attached to anamine group on a modified single-stranded DNA (2,21).The derivitization of an amine-containing DNA with DTPAand with another polyaminopolycarboxylate (ethylenediaminetetraacetic acid) was earlier reported and used forbinding stable iron ions (11). DTPA and its derivativeshave been useful for labeling proteins such as antibodieswith 1111nand several other radionuclides (19), but theyhave been less successful for labeling with @Tcbecause ofpoor label stability (9). Since label stability was a majorconcern of this investigation, the SHNH moiety was used asan alternative to DTPA for labeling DNA with 99mTc.TheSHNH moiety was developed for labeling antibodies with

@Tc(10); this laboratoryhas demonstratedthat,whenbound to antibodies by the SHNH moiety, @Tcdisplaysacceptable stability in vitro and in vivo (16). Accordingly,the SHNH moiety was considered herein for DNA labeling.

As shown in Figure 2, the primaiy amino groups attachedto the oligonucleotides of this study were readily conjugated with the NHS derivative of SHNH. Following conjugation, the UV peak of the unmodified DNA virtuallydisappeared on reversed-phase HPLC analysis of the conjugated oligonucleotide. In its place appeared another peakat greater retention time. The results with DTPA are identical except that, in this case, the conjugated DNA elutedearlier than the unmodified DNA. Whereas in past studiesfrom this laboratoiy of antibody conjugation with SHNH,the presence of high molecular weight protein aggregateswas a concern (16), no evidence of DNA aggregation wasobserved in this investigation. The concern in the use of thecyclic anhydride of DTPA is not aggregation but crosslinkingthroughthe twoanhydridegroups(19).As showninFigure 5, however, DNA dimers or oligomers, which would

100@

0111 In75

99m-@.% . Te

z 5Q@0

@ 0 25

TABLE IBiodistribution Results in Normal Mice 2.5 Hours

Posthtravenous Administration of Technetium-99m andIndium-i11-Labeled C-chains

Liver0.390.952.50.58Heart0.030.010.540.12KidneysI

.30.215.61.4Lung0.080.030.870.23Stomach0.210.392.50.67Spleen0.10.030.970.27Musde0.040.020.240.08Skin0.050.010.630.17Intestines0.10.111.41.7Blood0.08

(n=6)0.011.8

(n=6)0.37

0 6 16 24

TIME (hours)

FIGURE7. Percentagedissociationof @Tcand1111nfromIabaled D-chain bound to magnetic beads duting 37°Cincubation infresh human serum for up to 24 hr. Errorbars represent I s.d. of themean (n = 5 at all data points).

Forthe 1111n-labeledC-chain,althoughbindingto serumproteins was not observed (Fig. 6), a second peak corresponding to lower molecular weights was seen after 1 hr inserum and which became the prominent peak at 24 hr.Because 1111nlabeled to proteins by DTPA has been shownto be stable during serum incubations (19), the position ofthis peak strongly suggests degradation, possibly of theDNA phosphodiester-backboneby nucleases.

MimaI BlodistñbutionStud@sThe biodistribution at 2.5 hr postadministration to nor

mal mice are presented in Table 1 as the percentage ofinjected @Tcand 1111n.Important differences betweenlabels are readily apparent. The levels of @“Tcin all tissueswere significantly higher than 1111n(p < 0.001, Student'sunpaired t-test). A partial explanation for these differencesmost probably is related to the differences in serum proteinbinding for the two injectates; as demonstrated above (Fig.6), only the @Tc-DNAbinds to serum proteins. Thiswould raise activity levels in blood and, by virtue of theblood content of tissues, raise activity levels in tissues aswell. From the percentage of organ weight due to blood(20) and from the known blood activity, it is possible toestimate that less than 15% of the activity levels in tissue ofTable 1 (other than heart and spleen) were due to the bloodpool. The differences between labels may therefore be aconsequence of other factors such as instability of the @1InDNA to degradation in serum.

DISCUSSiONThe goal of this study was to develop a method of

labeling single-stranded DNAS with @“@Tcwhich would be

2312 The Journal of Nuclear Medicine •Vol. 36 •No. 12 •December 1995

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be readily apparent in this analysis, are not evident. Finally,there is little likelihood that treatment with either NHS orcyclic anhydride would derivatize functional groups of thebases themselves (11,22).

The SHNH-DNAs were radiolabeled with @mTcfollowing an approach essentially identical to that used routinelyfor the labeling of SHNH-conjugated antibodies with thisradionuclide (10, 16). No attempt was made to maximizelabeling efficiency, yet typical values were 40%—60%with

@‘Tc.Resultsimprovedwiththesubstitutionof tricineforglucoheptonate as transchelator (17). Labeling at roomtemperature proceeded more rapidly and the specific activities achievable appeared to be much higher.

In this study, the determination of labeling efficiency andlabel stability was aided by the use of oligonucleotidesderivatized with biotin. The peak @‘@Tcand “Inactivity inthe radiochromatographic profiles obtained by size-exclusion HPLC (Fig. 4) and the migration pattern on gel electrophoresis (Fig. 5) both showed a distinct shift toward ahigher molecular weight in the presence of streptavidin.The fact that a similar shift was not evident when biotinsaturated streptavidin was added conclusively demonstrated that the label was on the oligonucleotide. The comparable shift observed when the biotin-saturated,streptavidin-bound complementary chain was added is further evidence. As shown in Figure 7, the biotin moiety wasalso useful in demonstrating serum stability with thestreptavidin-conjugated beads.

That the shifts discussed above were not complete in anycase is probably an indication that the unpurified oligonucleotide preparations contained nonbiotinylated DNAchains. For instance, the oligonucleotides used in this studymay have been contaminated with a variety of DNA specieswith different chain lengths or missing the biotin moiety.Polyacrylamide electrophoresis studies (Fig. 5) have clearlyshown the presence of lower molecular weight contaminating DNA in preparations of A- and B-chains.

The stability of a label in serum is among the mostimportant factors for an agent under consideration for invivouse.Becauseof the highstabilityof @“@Tcon antibodies facilitated by the SHNH moiety (16 ), it may not besurprising that @Tcshowed only 4% dissociation fromDNA when incubated on beads in serum (Fig. 7). Moremeaningful, perhaps, is the stability in serum of the labelwhen incubated in solution. The rapid serum protein binding of @Tc-DNMobserved in this study, however, interfered with the determination of stability in that fashion.Nevertheless, some measure of serum stability is evident inthat the 99mTclabel was bound, at least in part, to serumproteins by DNA rather than in some “free―chemical formof 99mTc This is clear from the large percentage of labelwhich could be prevented from binding to proteins by theaddition of calf thymus DNA and which could be displacedby streptavidin-conjugated beads.

It is instructiveto speculate on the mechanismof 99mTc..DNA binding to serum proteins. It is tempting to suggestthat the binding may be related to the lipophilicity of the

6-member methylene linker by which the amine is conjugated to DNA. An identical DNA chain but with a hydroxyl-modified 3-carbon linker, however, was also used in thisresearch. Even though this linker is considerably less lipophilic, the serum binding properties of the @“@Tc-labeledoligonucleotide remained unchanged (data not presented).The possibility also exists that the binding may be related tothe base sequences selected for this investigation. The sameDNA chain, however, showed no tendency towards serumprotein binding when labeled with “In(Fig. 4). Finally, analternative explanation exists because the SHNH moietydoes not, in itself, satisfy the chelation requirements ofreduced 99mTc Since the complex must be “capped―withglucoheptonate or other species (10), it is possible thatserum proteins may participate in this process with theresult that the label would be seen to bind to serum proteins. The nature of the binding will need to be established.

Although serum protein binding was not observed for the“In-labeledC-chain, evidence of degradation upon serumincubation was observed only in this case (Fig. 6). Degradation by nucleases of oligonucleotides with unprotectedphosphodiester backbones, such as that used in this research, is a common observation (1 ). That the @mTc@la@beled C-chain shows no evidence of labeled degradationproducts during serum incubation may be a consequence ofthe serum protein binding.

CONCLUSIONOligonucleotides derivatized with a primaiy amine can

be conjugated with SHNH and radiolabeled stably with99mTc The stability of the label is comparable to thatobserved by us for “Inradiolabeled to the same oligonucleotides by DTPA. One interesting observation from thiswork is that @Tc-DNA,when labeled in this fashion, bindsto serum proteins. The binding is probably related to theSHNH moieties since similar binding was not observed for“In-DNA.A consequence of this binding was higher99mTcblood levels in vivo, but another consequence may beincreased stability to nucleases in serum since significantdegradation products were observed in this work only for“In.Furtherinvestigationsshouldestablishwhetherserum binding of @“Tc-DNMwill interfere with targeting invivo.

ACKNOWLEDGEMENTS

The authorsthank Dr. MichaelAbrams,JohnsonMattheyInc.,for providing the NHS-SHNH used in this investigation and Ms.Eleni Millona and Ms. Sophia Kim for their assistance in thecompletionof the manuscript.Thisworkwassupportedin part byDE-FGO2-93ER61656 from the U.S. Department of Energy andby CA59785 from the National Cancer Institute.

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2314 The Journal of NuclearMedicine•Vol.36 •No. 12 •December 1995

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1995;36:2306-2314.J Nucl Med.   D.J. Hnatowich, P. Winnard, Jr., F. Virzi, M. Fogarasi, T. Sano, C.L. Smith, C.R. Cantor and M. Rusckowski  Technetium-99m Labeling of DNA Oligonucleotides

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