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Technetium-99m Radiolabeling: Oligonucleotide Binding and Label Stability Study, Lecture notes of Engineering

A method for radiolabeling DNA with Technetium-99m using a derivative of DTPA. The study investigates the stability of the label in serum and its biodistribution in normal mice. The results show that the label was bound to serum proteins, and the stability of the label in serum was determined. The document also discusses the properties of the labeled DNAs and their behavior in serum.

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bg1
are used, either to block transcription of genes within the
nucleus of cells or to block translation of messenger RNA
within the cytoplasm (1,2). Accordingly, if radiolabeled,
these oligonucleotides may usefully carry radioactivity to
targeted cells or tissues. This and other applications of
these reagents as radiopharmaceuticals, however, will re
quire that methods be developed for radiolabeling them
with diagnostic and, possibly, therapeutic radionuclides.
Methods for radiolabeling oligonucleotides with beta-emit
ting radionuclides such as 3H,@ and 32P are well estab
lished (3—6);however, the use of oligonucleotides radiola
beled with imageable radionuclides is in its infancy.
Methods have been reported for labeling oligonucleotides
with ‘@I(6,7) and, as such, these methods should be useful
with ‘@Iand ‘@‘Iradionuclides with imaging properties.
Depending on the pharmacokinetic properties of an oligo
nucleotide of interest, it is likely that the imaging radionu
clide of choice will often be 99mTc Recently, a method for
radiolabeling DNA with @Tcwas described which relies
upon a derivative of DTPA attached to the oligonucleotide
to form chelates with reduced @‘@‘Tc(8). DTPA has previ
ously been used for radiolabeling antibodies with @“Tcbut
was abandoned when the instability of the label wasjudged
to be unacceptably high (9). Accordingly, this study was
performed to investigate an alternative approach to label
oligonucleotides stably with @Tc.A hydrazino nicoti
namide (SHNH) moiety has been shown to form stable
complexes with @Tcwhen conjugated to antibodies (10).
In this investigation, an amine-derivatized DNA was con
jugated with SHNH and the properties of the label evalu
ated. To provide a useful comparison, the same DNA was
alsoconjugatedwithDTPA,ashasbeendescribedbyoth
ers (2,11), for radiolabeling with “Inusing procedures
routine for antibody labeling (12).
In this investigation, 22-base, single-stranded DNASwere
selected because their small size (about 8 kDa) was ex
pected to facilitate rapid whole-body clearance after admin
istration. Moreover, a 22-base oligonucleotide is large
enough to virtually exclude any possibility of an accidental
match within the genomic DNA and its transcripts (1).
DNAS used were derivatized on one end with a biotin
moiety to provide a useful means of establishing labeling
efficiency and label stability. Using this feature, radiola
beled DNAS may be easily distinguished from unbound or
@ngIe-strandedRNAand DNA oligonucleotides may be useful
as radiopharmaceuticais for antisense and other in vivo appli
cations if convenient methods for stably attaching radionuclides
such as @‘Tccan be developed. Methods: To radiolabel DNA
with @‘1c,we have used the hydrazino nicotinamkle (SHNH)
rnolety devebped elsewhere. The diethylenetriaminepenta
acetic acid (DWA) chelate was used to label DNA with 111lnfor
comparison. Complementary 22-base, single-stranded oligonu
cleotides were obtained, each wfth a pnmary amine attached to
eitherthe 3' or 5' end and witha biotinmoietyonthe opposite
end. The DNA was conjugated with SHNH by a N-hydroxy
succinimide derivative and with DTPA by the cyclic anhydride.
Results: Reversed-phase HPLCanalysis showed that essen
tially complete conjugation was achieved in both cases. The
purifiedSHNH-DNAwas radiolabeledwith @rcby transche
lation 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 streptavidin
through the biotin moietiesand to hybridIzewiththeir compie
mentary DNAin saline was retained for both radiolabels as
determined by size-exclusion HPLC analysis. HPLC radiochro
matograms of serum incubates showed a shift of @‘@‘Tc,but not
1111n,to a high molecular weight, strongly suggesting serum
protein binding in the fomier case only. Low-molecular wsight
degradation products were seen with ‘11ln,but not with @Tc
and may be related to the use of phosphodiester-linked oligo
nucleotides. As a further measure of label stability, the DNAs
were bound to strepta@Adin-conjugated magnetic beads and
incubated in fresh 37°Chuman serum. Less than 4% of @Fc
and 14% of 1111nwas lost in 24 hr. Conclusion: Amino-modi
fled,single-stranded DNAcan be stablyradiolabeledwith @1c
by the SHNHmoietywithout loss of function.
KeyWords oligonucleotides;technetium-99m;radiolabeling
J NuciMed1995362306-2314
he extraordinary properties of DNA and RNA suggest
that there is potential for the use of these oligonucleotides
as radiopharmaceuticals. For example, there is current in
terest in antisense applications in which oligonucleotides
ReceivedAug.4,1994;revisionacceptedDec.29,1994.
For correspondence or reprints contact: D.J. HnatOWich,PhD, Department of
NudearMedicine,UniversftyofMassachUsettsMed@alCent&,Worcester,MA
01655.
2306 The Journal of Nuclear Medicine•Vol.36 •No. 12 •December 1995
Technetium-99m Labeling of DNA
Oligonucleotides
D.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 Advanced
Biotechnology and Departments of Biomedical Engineering Phartnacolo@jiand Biology,Boston University, Boston, Massachusetts
pf3
pf4
pf5
pf8
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are used, either to block transcription of genes within the

nucleus of cells or to block translation of messenger RNA

within the cytoplasm (1,2). Accordingly, if radiolabeled, these oligonucleotides may usefully carry radioactivity to

targeted cells or tissues. This and other applications of

these reagents as radiopharmaceuticals, however, will re

quire that methods be developed for radiolabeling them

with diagnostic and, possibly, therapeutic radionuclides.

Methods for radiolabeling oligonucleotides with beta-emit

@ ting radionuclides such as 3H, and 32P are well estab

lished (3—6);however, the use of oligonucleotides radiola

beled with imageable radionuclides is in its infancy.

Methods have been reported for labeling oligonucleotides

with ‘@I(6,7) and, as such, these methods should be useful

with ‘@Iand ‘@‘Iradionuclides with imaging properties.

Depending on the pharmacokinetic properties of an oligo

nucleotide of interest, it is likely that the imaging radionu

clide of choice will often be 99mTc Recently, a method for

radiolabeling DNA with @Tcwas described which relies

upon a derivative of DTPA attached to the oligonucleotide

to form chelates with reduced @‘@‘Tc(8). DTPA has previ

ously been used for radiolabeling antibodies with @“Tcbut

was abandoned when the instability of the label was judged

to be unacceptably high (9). Accordingly, this study was

performed to investigate an alternative approach to label

oligonucleotides stably with @Tc.A hydrazino nicoti

namide (SHNH) moiety has been shown to form stable

complexes with @Tcwhen conjugated to antibodies (10).

In this investigation, an amine-derivatized DNA was con

jugated with SHNH and the properties of the label evalu

ated. To provide a useful comparison, the same DNA was

alsoconjugatedwithDTPA,as hasbeendescribedbyoth

ers (2,11), for radiolabeling with “Inusing procedures

routine for antibody labeling (12).

In this investigation, 22-base, single-stranded DNASwere

selected because their small size (about 8 kDa) was ex

pected to facilitate rapid whole-body clearance after admin

istration. Moreover, a 22-base oligonucleotide is large

enough to virtually exclude any possibility of an accidental

match within the genomic DNA and its transcripts (1).

DNAS used were derivatized on one end with a biotin

moiety to provide a useful means of establishing labeling

efficiency and label stability. Using this feature, radiola

beled DNAS may be easily distinguished from unbound or

@ngIe-strandedRNA and DNA oligonucleotides may be useful

as radiopharmaceuticais for antisense and other in vivo appli

cations ifconvenient methods for stably attaching radionuclides such as @‘Tccan be developed. Methods: To radiolabel DNA with @‘1c,we have used the hydrazino nicotinamkle (SHNH)

rnolety devebped elsewhere. The diethylenetriaminepenta

acetic acid (DWA) chelate was used to label DNAwith 111lnfor comparison. Complementary 22-base, single-stranded oligonu cleotides were obtained, each wfth a pnmary amine attached to

either the 3' or 5' end and witha biotinmoietyon the opposite

end. The DNA was conjugated with SHNH by a N-hydroxy succinimide derivative and with DTPA by the cyclic anhydride.

Results: Reversed-phase HPLCanalysis showed that essen

tially complete conjugation was achieved in both cases. The

purifiedSHNH-DNAwas radiolabeledwith @rcby transche

lation 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 streptavidin

through the biotinmoieties and to hybridIzewiththeir compie

mentary DNA in saline was retained for both radiolabels as

determined by size-exclusion HPLC analysis. HPLC radiochro matograms of serum incubates showed a shift of @‘@‘Tc,but not

1111n,to a high molecular weight, strongly suggesting serum

protein binding in the fomier case only. Low-molecular wsight degradation products were seen with ‘11ln,but not with @Tc and may be related to the use of phosphodiester-linked oligo nucleotides. As a further measure of label stability, the DNAs were bound to strepta@Adin-conjugated magnetic beads and incubated in fresh 37°Chuman serum. Less than 4% of @Fc

and 14% of 1111nwas lost in 24 hr. Conclusion: Amino-modi

fled,single-stranded DNAcan be stably radiolabeledwith @1c

by the SHNHmoietywithoutloss of function.

Key Words oligonucleotides;technetium-99m;radiolabeling

J NuciMed1995362306-

he extraordinary properties of DNA and RNA suggest

that there is potential for the use of these oligonucleotides

as radiopharmaceuticals. For example, there is current in

terest 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,MA

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

Technetium-99m Labeling of DNA

Oligonucleotides

D.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 Advanced Biotechnology and Departments of Biomedical Engineering Phartnacolo@jiand Biology, Boston University, Boston, Massachusetts

0

@ 0 00 BrnN @ oxo,_@

FiGURE1. Structureof A and B DNA

chains used in this investigation.

dissociated label by size-exclusion analysis before and after

the addition of streptavidin (13).

In this study, we describe the results of conjugating 5ev

eral single-stranded DNAS with SHNH and DTPA fol

lowed by labeling with @Tcand ‘‘‘In,respectively. The

stability of the label was determined in serum, and biodis tribution 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.The

base sequences were 5'-biotinTA ATA CGA CFC ACF ATA GGG AGamine-3' (A-chain) and 5'-amineGG TAC AGG TCf

CAC TGT ATG ACbiotin(C-chain)and their complements(B-

and D-chains, respectively). Figure 1 presents the structure of the

A- or B-chain (i.e., with the biotin moiety on the 5' end). As

shown, the biotin moiety was attached directly through a 15- member amide-polyether linker to the terminal phosphate, while the amine was normally attached to the terminal phosphate group through a 6-member methylene carbon spacer. The molecular weight of each chain was about 8.2 kDa. The melting and anneal

ing temperatures in physiologicalsaline were calculated to be

57—60°Cand 32—41°C,respectively, for both pairs (14). The DNAS were purchased unpurified and were used without further purifi cation. They were generally handled under sterile conditions; all solutions were sterilized by terminal filtration through a 0.22-sm filter, and sterile pipette tips were used. All other pipette tips and tubes were autoclaved prior to use. Streptavidin was purchased and used without further purifica tion. The DNAs were stored dry as received, at refrigerator tern peratures and were dissolved at a concentration of 1—4mg/ml in sterile water when needed. After solubilization, 20—500p.g DNA

were added to sterile plasticvialswhichwere immediatelyfrozen

for storage. Technetium-99m-pertechnetate was obtained from a @Mo@@mTcradionuclide generator and ‘‘‘Inwas purchased as the chloride complex. Streptavidin-conjugated superparamagnetic polystyrene beads (Dynabeads M-280, Dynal, A.S., Lake Success, NY) were stored at refrigerator temperatures as recommended by the manufacturer. The N-hydroxysuccinimide (NHS) derivative of the hydrazino nicotinamide moiety was a gift from Dr. M. Abrams,

(JohnsonMattheyInc., West Chester, PA). The cyclicanhydride

of DTPA was synthesized as previously described (15).

Oligonucleotide Conjugation

In this investigation,all four DNAchainswere derivatizedwith

either SHNH or DTPA. In the former case, the NHS-derivative of SHNH moiety was dissolved in dry dimethylformamide to a con centration of 8.6 mg/ml (10). For conjugation, the DNA solution

was diluted with a sterile bicarbonate buffer so that the final

concentrations were 2.0 mg/ml DNA, 1 M NaCl, 0.25 M NaHCO3, 1.0 mM EDTA at pH 8.3—9.0.The EDTA was added to complex cations such as calcium. The DNA solutions were incubated for 30

mmat 45°Cjustprior to SHNHadditionto dissociateanysecond

ary complexes between the primary amines and the phosphate

backbone.Between0.2 to 1 mgof DNAwasconjugatedat a 1—

molarexcessof SHNHto DNAbyaddingthe necessaryvolumeof

dimethylformarnidesolutiondropwiseto the DNA solutionwhile

vortexing.The solutionwas incubatedat room temperature for 1

hr. Followingincubation,the conjugatedoligonucleotidewas pu

rifled on a 0.7 x 20 cm gel filtration column of P-4 using a sterile 0.25 M ammonium acetate, 1.0 mM DTPA buffer at pH 5.2 as eluant. The DTPA was added to complex excess stannous ion and help prevent radiocolloid formation. Fractions (0.4 ml) were col lected and the absorbance (260 nm) of each measured. Oligonu

cleotideconcentrationswere estimatedusingan extinctioncoeffi

cient determined in this laboratory of 30 @.d/@gfor a 0.1% solution

measuredat 260nm.The absorbanceof SHNHat thiswavelength

and under these conditions was found to be insignificant. The fractions collected contained DNA at concentrations in the 0.5—1.

mg/mirange.

ConjugationwithDTPAwasachievedwiththe cyclicanhydride

as previously described for proteins (12). Approximately 1 mg of the oligonucleotide was dissolved in 10 ml 0.4 M HEPES buffer, pH 8.5, and this solution was added rapidly to a round-bottom test tube containing 100 mg of the thy anhydride while vortexing. Thus,

the DTPA:DNAmolar ratio was 2110:1.The solutionwas incu

bated at room temperature for 30 mm. The conjugatedoligonu

cleotides were originally purified on a gel-filtration column of P- using 10% ethanol as an eluant, but the inability to remove an

unidentifiedradiochemicalcontaminantusingthis columnneces

sitatedthe use of 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,

IC) c\

z

Cl)

@ 0.

RESULTS

Oligonucleotide Conjugation

In this study, the amine-derivatized DNAS were conju

gated with an NHS derivative of SHNH and with the cyclic

anhydride of DTPA. Figure 2 presents UV absorption chro

matograms obtained by reversed-phase HPLC of unmodi

fled A-chain (panel 1), SHNH-conjugated A-chain prior to

purification (panel 2), and DTPA-conjugated A-chain after

purification (panel 3). The unmodified A-chain is clearly

resolved into one major peak at a retention time of about

12 mm and several minor peaks.

Conjugation with SHNH was accomplished at molar ra

tios of SHNFI:DNA of between 1—25:1.No differences in

the ability of the conjugated oligonucleotide to hybridize or

to accept a @Tclabel were observed at these molar ratios

(data not presented). Accordingly, all subsequent conjuga

tions were performed at a 1:1 molar ratio. After conjugation with either SHNH or DTPA, the pres

ence of unmodified A-chain was reduced to a minor con

stituent (Fig. 2). The peak absorbance shifted to 21 mm in

the SHNH case and 3 mm in the DTPA case. The unmod

ified DNA peak is essentially absent, demonstrating that

conjugation was largely complete.

Because DTPA-coupled oligonucleotides purified on a

P-2 column contained unacceptably high levels of uniden

rifled radiochemical contaminant(s) after radiolabeling; pu

rification was ultimately achieved on an anion exchange

column. Figure 3 shows the UV absorbance profiles of

fractions off this column. The high acidity (pH 2.7) of the initial eluant apparently results in complete protonation of

free DTPA which then elutes from the column early. The

DNA, however, still possesses a high negative charge

through its phosphate groups and is retained. As the ionic

strength is increased, however, the increasing sodium ion

concentration apparently neutralizes this charge and per

mits the DNA to elute. The three major peaks (eluting at

20, 100 and 150 ml in the figure) were analyzed by gel

TIME (mm.)

FiGURE2. UV absorptionchromatogramsobtained by re

versed-phase HPLC analysis of unmodified A-chain (trace 1), of SHNH-conjugated A-chaln prior to purification (trace 2) and of DTPA-conjugated A-chaln prior to puiiflcation(trace 3).

washed three times with the washing buffer. The serum, the com bined washes and the beads were then counted in a NaI(Tl) well counter.

AnimalBiodistñbutionShidues Biodistributions of both @“Tc-and ‘DIn-labeledD-chain were determined in normal CD-i male mice. Each animal received by

tail vein administration0.1 ml of saline containingeither 7.5 @g

@ (7.3 MCi)“In-or 10 (7 MCi)99mTc..labeledDNA. Animals

were killed by spinal dislocation 2.5 hr postadministration. Sam ples of organs were rinsed in cold saline and were counted along

with a blood sampleand an aliquot of the injectatein a NaI(Tl)

wellcounter.The biodistributionswerereported as the percentage

of administered radioactivity per gram of tissue.

vol. (ml) 0— 50— 144 145—

1% HAc, pH 2. 17@NH 4Ac. pH 5. 1% HAc, pH 2.

0.

0.

@ 0.3^ z

a 0

c,i

FiGUREa uv absorptionprofileof free

tions obtained by anion exchange chroma tography of DTPA-conjugatedC-chsin. Ofthe three major peaks (at 20, 100 and 150 ml), only the latter was shown to contain DNk

@@ :: r\ @: /

@@ :@ @JJ@@/

@ 0.0 --/@.

0 20 40 60 80 100 120 140 160 150 VOLUME (ML)

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

FIGURE 4. Radiochromatograms ob

talned by size-exclusionHPLCanalysis of

99mTc@labeiedC-chain (leftcolumn)and 111ln labeled C-chain (right column) in saline (trace 1), in saline containing biotin-saturated streptavidin (trace 2), in saline containing streptavidin (trace 3) and in saline containing complementary D-chain bound to streptavi din (trace 4).

electrophoresis. Only the latter peak contained DNA (see

below). The recovery of DNA in this purification was 16%.

Oligonucleotide Labeling

@ When 100 or more of the SHNH-conjugated DNAS at

a concentration of at least 250 @tWmIwere radiolabeled with

up to 1 mCi @°@Tc,labeling efficiencies ranged between

30%—60%,as determined by Sephadex 0-50 chromatogra

phy. Attempts to label smaller amounts of DNA or at lower

concentrations resulted in reduced efficiencies. These ob

servations were independent of the DNA chain. As con trols, the unmodified DNAS were radiolabeled in the iden tical fashion. Labeling efficiencies were less than 5% in the case of each 99mTc control. When the @“Tc-labeledoligo nucleotides were analyzed by size-exclusion HPLC, a single

peak was always observed. Recoveries occasionally ap

proached 100%, although typical recoveries were 81% ±

5% (s.d., n = 4). That recoveries could be increased by increasing the salinity of the eluant suggests that this reten

tion was due, in part, to ionic interactions of the charged

DNAS with the column support.

Typically, specific activities of 50 @Ci4tgDNA were

achieved when glucoheptonate was replaced with tricine. Properties of the labeled DNAS such as biotin binding,

hybridization, protein binding in serum or serum stability

appeared to be unchanged with respect to DNA labeled via

glucoheptonate.

Labeling efficiencies of the DTPA-conjugated and

DEAE-purified oligonucleotides approached 100% under

most conditions of DNA concentration and at specific ac

0 U)

tivities of 60 @Ci/.tg.The unconjugated C-chain under

identical conditions retained only 2% of the “Inlabel.

Figure 4 presents size-exclusion HPLC radiochromato

grams of C-chain radiolabeled with 99mTc(left column) and

“In(rightcolumn)before(trace1) andafter(trace3) the

addition of excess streptavidin. Both labeled DNAS elute in

a single peak which largely shifts to a higher molecular

weight (i.e., smaller elution volumes) with the addition of

the streptavidin. Similar shifts to high molecular weight

were observed with the addition of the streptavidin-D-chain

(complementary) construct (trace 4). Also in Figure 4 are

radiochromatograms showing no shift with the addition of

biotin-saturated streptavidin (trace 2). Recoveries in these

HPLC analyses were always 80%—90%.Because

[ @TcJpertechnetateisretained,thegoodrecoveriesdem

onstrate that oxidation of the label to pertechnetate was not

an important mode of instability.

Elecfro@ Shidies

The first four lanes in the electrophoretogram of Figure

5 contain unmodified DNA chains A through D, respec

tively, stained with ethidium bromide. Whereas chains C

and D show only one band at the position expected for a

22-base DNA, that of chains A and B show two distinct

bands, one co-migrating with chains C and D plus an addi

tional band with an apparently lower molecular weight.

Several other minor bands corresponding to higher molec

ular weight have occasionally been seen as well. Lanes 5—

contain aliquots of three peak fractions (at 20, 100 and 150

ml, respectively) from the DEAE anion exchange purifica

99m 111 In

ELUTION VOLUME

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

@@TcOrgan 111ln

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

Resultspresented as percent injecteddose per gram (% big) with

s.d.ofthemean.Thelevelsof @1cinalltissuesaresignificanflyhigher

than 111ln(p < 0.001, Student's unpairedt-test).

useful for in vivo imaging applications. Recently, a method

of radiolabeling oligonucleotides has been reported in

which a derivative of DTPA is covalently attached to an

amine group on a modified single-stranded DNA (2,21).

The derivitization of an amine-containing DNA with DTPA

and with another polyaminopolycarboxylate (ethylenedia

minetetraacetic acid) was earlier reported and used for

binding stable iron ions (11). DTPA and its derivatives

have been useful for labeling proteins such as antibodies

with 1111nand several other radionuclides (19), but they

have been less successful for labeling with @Tcbecause of

poor label stability (9). Since label stability was a major concern of this investigation, the SHNH moiety was used as

an alternative to DTPA for labeling DNA with 99mTc.The

SHNH moiety was developed for labeling antibodies with @Tc(10); this laboratoryhas demonstratedthat, when bound to antibodies by the SHNH moiety, @Tcdisplays

acceptable 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 attached

to the oligonucleotides of this study were readily conju

gated with the NHS derivative of SHNH. Following conju

gation, the UV peak of the unmodified DNA virtually

disappeared on reversed-phase HPLC analysis of the con

jugated oligonucleotide. In its place appeared another peak

at greater retention time. The results with DTPA are iden

tical except that, in this case, the conjugated DNA eluted

earlier than the unmodified DNA. Whereas in past studies

from this laboratoiy of antibody conjugation with SHNH,

the presence of high molecular weight protein aggregates

was a concern (16), no evidence of DNA aggregation was

observed in this investigation. The concern in the use of the

cyclic anhydride of DTPA is not aggregation but cross linkingthroughthe twoanhydridegroups(19).As shownin

Figure 5, however, DNA dimers or oligomers, which would

100@

0111 In 75

99m

-@.%. Te

z 5Q@ 0

TABLE I

Biodistribution Results in Normal Mice 2.5 Hours Posthtravenous Administration of Technetium-99m and

Indium-i11-Labeled C-chains

Liver0.390.952.50.58Heart0.030.010.540.12KidneysI

.30.215.61.4Lung0.080.030.870.23Stomach0.210.392.50.67Spleen0.10.030.970.27Mus

(n=6)0.

(n=6)0.

TIME (hours)

FIGURE7. Percentagedissociationof @Tcand 1111nfromIa

baled D-chain bound to magnetic beads duting 37°Cincubation in fresh human serum for up to 24 hr. Errorbars represent I s.d. of the mean (n = 5 at all data points).

Forthe 1111n-labeledC-chain,althoughbindingto serum

proteins was not observed (Fig. 6), a second peak corre

sponding to lower molecular weights was seen after 1 hr in

serum and which became the prominent peak at 24 hr.

Because 1111nlabeled to proteins by DTPA has been shown

to be stable during serum incubations (19), the position of

this peak strongly suggests degradation, possibly of the

DNA phosphodiester-backboneby nucleases.

MimaI BlodistñbutionStud@s

The biodistribution at 2.5 hr postadministration to nor

mal mice are presented in Table 1 as the percentage of

injected @Tcand 1111n.Important differences between

labels are readily apparent. The levels of @“Tcin all tissues

were significantly higher than 1111n(p < 0.001, Student's

unpaired t-test). A partial explanation for these differences

most probably is related to the differences in serum protein

binding for the two injectates; as demonstrated above (Fig.

6), only the @Tc-DNAbinds to serum proteins. This

would raise activity levels in blood and, by virtue of the

blood content of tissues, raise activity levels in tissues as

well. From the percentage of organ weight due to blood

(20) and from the known blood activity, it is possible to

estimate that less than 15% of the activity levels in tissue of

Table 1 (other than heart and spleen) were due to the blood

pool. The differences between labels may therefore be a

consequence of other factors such as instability of the @1In

DNA to degradation in serum.

DISCUSSiON

The 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

be readily apparent in this analysis, are not evident. Finally,

there is little likelihood that treatment with either NHS or

cyclic anhydride would derivatize functional groups of the

bases themselves (11,22).

The SHNH-DNAs were radiolabeled with @mTcfollow

ing an approach essentially identical to that used routinely

for the labeling of SHNH-conjugated antibodies with this

radionuclide (10, 16). No attempt was made to maximize

labeling efficiency, yet typical values were 40%—60%with

@‘Tc.Resultsimprovedwiththe substitutionof tricinefor glucoheptonate as transchelator (17). Labeling at room

temperature proceeded more rapidly and the specific activ

ities achievable appeared to be much higher.

In this study, the determination of labeling efficiency and

label stability was aided by the use of oligonucleotides

derivatized with biotin. The peak @‘@Tcand “Inactivity in

the radiochromatographic profiles obtained by size-exclu

sion HPLC (Fig. 4) and the migration pattern on gel elec

trophoresis (Fig. 5) both showed a distinct shift toward a

higher molecular weight in the presence of streptavidin.

The fact that a similar shift was not evident when biotin

saturated streptavidin was added conclusively demon

strated that the label was on the oligonucleotide. The com

parable shift observed when the biotin-saturated,

streptavidin-bound complementary chain was added is fur

ther evidence. As shown in Figure 7, the biotin moiety was

also useful in demonstrating serum stability with the

streptavidin-conjugated beads.

That the shifts discussed above were not complete in any

case is probably an indication that the unpurified oligonu

cleotide preparations contained nonbiotinylated DNA

chains. For instance, the oligonucleotides used in this study

may have been contaminated with a variety of DNA species

with different chain lengths or missing the biotin moiety.

Polyacrylamide electrophoresis studies (Fig. 5) have clearly shown the presence of lower molecular weight contaminat ing DNA in preparations of A- and B-chains.

The stability of a label in serum is among the most

important factors for an agent under consideration for in

vivouse. Becauseof the highstabilityof @“@Tcon antibod ies facilitated by the SHNH moiety (16 ), it may not be surprising that @Tcshowed only 4% dissociation from

DNA when incubated on beads in serum (Fig. 7). More

meaningful, perhaps, is the stability in serum of the label

when incubated in solution. The rapid serum protein bind

ing of @Tc-DNMobserved in this study, however, inter

fered with the determination of stability in that fashion.

Nevertheless, some measure of serum stability is evident in

that the 99mTclabel was bound, at least in part, to serum

proteins by DNA rather than in some “free―chemical form

of 99mTc This is clear from the large percentage of label

which could be prevented from binding to proteins by the

addition of calf thymus DNA and which could be displaced

by streptavidin-conjugated beads.

It is instructiveto speculate on the mechanismof 99mTc..

DNA binding to serum proteins. It is tempting to suggest

that the binding may be related to the lipophilicity of the

6-member methylene linker by which the amine is conju

gated to DNA. An identical DNA chain but with a hydrox

yl-modified 3-carbon linker, however, was also used in this

research. Even though this linker is considerably less li

pophilic, the serum binding properties of the @“@Tc-labeled

oligonucleotide remained unchanged (data not presented).

The possibility also exists that the binding may be related to

the base sequences selected for this investigation. The same

DNA chain, however, showed no tendency towards serum

protein binding when labeled with “In(Fig. 4). Finally, an

alternative explanation exists because the SHNH moiety

does not, in itself, satisfy the chelation requirements of

reduced 99mTc Since the complex must be “capped―with

glucoheptonate or other species (10), it is possible that serum proteins may participate in this process with the result that the label would be seen to bind to serum pro teins. 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 serum

incubation was observed only in this case (Fig. 6). Degra

dation by nucleases of oligonucleotides with unprotected

phosphodiester backbones, such as that used in this re

search, is a common observation (1 ). That the @mTc@la@

beled C-chain shows no evidence of labeled degradation

products during serum incubation may be a consequence of

the serum protein binding.

CONCLUSION

Oligonucleotides derivatized with a primaiy amine can

be conjugated with SHNH and radiolabeled stably with

99mTc The stability of the label is comparable to that

observed by us for “Inradiolabeled to the same oligonu

cleotides by DTPA. One interesting observation from this

work is that @Tc-DNA,when labeled in this fashion, binds

to serum proteins. The binding is probably related to the

SHNH moieties since similar binding was not observed for

“In-DNA.A consequence of this binding was higher

99mTcblood levels in vivo, but another consequence may be increased stability to nucleases in serum since significant

degradation products were observed in this work only for

“In.Furtherinvestigationsshouldestablishwhetherse

rum binding of @“Tc-DNMwill interfere with targeting in

vivo.

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 the

completionof the manuscript.Thisworkwassupportedin part by

DE-FGO2-93ER61656 from the U.S. Department of Energy and by CA59785 from the National Cancer Institute.

REFERENCES

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  1. DewanjeeMK.Radiolabeledantisenseprobes:diagnosisand therapy.Diag Oncoll993;3:189—208.

Labeling DNA Oligonucleotides with Technetium-99m •Hnatowich at al. 2313