Bioconjugate Chem. 1997, 8, 238−243
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Synthesis of 125I-Labeled Oligonucleotides from Tributylstannylbenzamide Conjugates Michael W. Reed,*,† Igor G. Panyutin,‡ Don Hamlin,§ Deborah D. Lucas,† and D. Scott Wilbur§ Epoch Pharmaceuticals Inc., 1725 220th Street S.E. No. 104, Bothell, Washington 98021, Department of Nuclear Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, and Department of Radiation Oncology, University of Washington, Seattle, Washington 98195. Received October 16, 1996X
A rapid and efficient method for the synthesis of 125I-labeled oligodeoxynucleotides ([125I]ODNs) is described. The key intermediates are tributylstannylbenzamide-modified ODNs (Sn-ODNs). Reaction conditions are described for the preparation of 5′-modified Sn-ODNs. Treatment with NaI and chloramine T gave conversion to the desired I-ODN, which was easily isolated by reversed phase chromatography. Thermal denaturation (Tm) studies showed that hybridization properties were not disturbed by the 4-iodobenzamide modification. An [125I]ODN was prepared and characterized by hybridization to 32P-labeled DNA targets. Sequence specific cleavage of the target DNA strand by 125I was measured.
INTRODUCTION 125 I-labeled oligodeoxynucleotides ([125I]ODNs)1 have been used for years as probes to detect complementary nucleic acids. Appropriately labeled [125I]ODNs can also act as efficient, sequence specific, DNA cleavage agents (1). The nuclear decay process of 125I (electron capture and internal conversion) generates ∼22 low-energy electrons that dissipate their energy within a few nanometers of the decay site (2). If this decay process (Auger emission) occurs in close proximity to duplex DNA, double-strand breaks can occur with ∼100% efficiency/ decay event. The short DNA cleavage range of the Auger emitting isotopes makes them promising agents for sequence specific targeting by oligonucleotides. For example, when 125I-labeled deoxycytidine ([125I]dC) was enzymatically incorporated into a single position in an ODN and hybridized to a complementary ssDNA target, more than 70% of the cleavage products were found within 15-20 Å of the site of 125I decay (1). Recently it has been shown that an 125I-dC labeled triplex forming ODN can specifically cleave a homopurine sequence in a plasmid DNA target containing the nef gene of HIV. Each 125I decay gave approximately 0.8 dsDNA breaks (3). As a result of its efficient DNA cleavage properties, 125I is extremely cytotoxic when incorporated into the DNA of dividing cells as [125I]dU (4). Therapeutic (anticancer) implications of Auger emitting radionuclides have been reviewed (5). Although 125I is an efficient DNA cleavage agent, its 60 day half-life is an obvious disadvantage for in vivo applications. The shorter half-life, Auger emitting isotope 123I (13.2 h half-life) has also been shown to be cytotoxic when incorporated into DNA using [123I]dU (6)
* Author to whom correspondence should be addressed [telephone (206) 485-8566; fax 206/486-8336; e-mail mreed@ epochpharm.com]. † Epoch Pharmaceuticals. ‡ National Institutes of Health. § University of Washington. X Abstract published in Advance ACS Abstracts, March 1, 1997. 1 Abbreviations: ODN, oligodeoxynucleotide; I-ODN, iodinated ODN; Sn-ODN, tributyltin-modified ODN; ssDNA, singlestranded DNA; dsDNA, double-stranded DNA; ChT, chloramine T (sodium salt of N-chloro-p-toluenesulfonamide); TEAA, triethylammonium acetate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.
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or targeted to DNA using 123I-labeled estrogens (7). The DNA cleavage efficiency of [123I]dU incorporated into cellular DNA has been determined (8). Each decay of 123I gave 0.45-0.74 dsDNA breaks if DNA repair is accounted for. Radiolabeling of ODNs with 123I has not been reported. This paper describes a rapid and convenient radiolabeling method for making I-ODNs from tributylstannylbenzamide-modified ODN intermediates (Sn-ODNs). Aryl tin intermediates have been valuable for radiolabeling small molecular weight compounds via halodestannylation reactions (9). A 15-mer ODN bearing a 5′terminal hexylamine linker was used to develop the conjugation chemistry. The effect of the 4-iodobenzamide linker system on hybridization properties of nontadioactive (127I) labeled ODNs was studied spectrophotometrically. The terminally modified [125I]ODN was then prepared and hybridized to a 32P-labeled ssDNA target. Analysis of the 32P fragments after 15 days showed sequence specific DNA cleavage at the site of the attached 125I. Our ultimate goal is to prepare 123I-labeled ODNs and to determine how the position of attachment of 125I and 123I affects DNA cleavage efficiency of I-ODNs. EXPERIMENTAL PROCEDURES
General Chemical Procedures. Reversed phase HPLC analysis of nonradioactive compounds used a Rainin pump system. Pump control and data processing used a Rainin Dynamax chromatographic software package (Macintosh). Aqueous solutions were dried at 95% pure by HPLC and one major band by PAGE. 5′-Modified Sn-ODN (2). To 0.13 mL of a 0.77 mM solution of ODN 1 (0.10 µmol) were added 0.1 mL of 1 M sodium borate buffer (pH 8.3) and 0.3 mL of 0.1 M sodium borate buffer (pH 8.3) in a 1.7 mL Eppendorf tube. A solution of 5 mg (10 µmol) of N-hydroxysuccinimidyl 4-tri(n-butyl)stannylbenzoate (13) in 0.5 mL of THF was added, and the milky emulsion was shaken for 16 h. The mixture was concentrated to a volume of ∼0.1 mL on a Speed Vac to remove THF. The cloudy solution was dissolved in 0.4 mL of 0.1 M TEAA (pH 7.5) and filtered through a 0.45 µm syringe filter. The filter was rinsed with an additional 0.2 mL of buffer, and the combined filtrate was purified by HPLC using the conditions described in Figure 2. The desired product (31 min peak) was collected, and 0.1 mL of 1 M borate buffer (pH 8.3) was added before taking to dryness on a Speed Vac. The white solid product was reconstituted with 0.5 mL of water, and the concentration was determined by A260 measurement. Recovery of 2 was 0.23 mg (42% yield). 5′-Modified [127I]ODN (3). Method A. To 12 µL of an 83 µM solution of Sn-ODN 2 (1 nmol) were added 7.5 µL (5 nmol) of a 0.1 mg/mL solution of sodium iodide and 6.9 µL of 0.1 M borate buffer (pH 8.3). Reaction was initiated by adding 4.56 µL (20 nmol) of a 1 mg/mL solution of chloramine T hydrate (ChT) in water. After 5 min, the reaction was quenched with 5 µL of a 10 mg/ mL solution of sodium bisulfite. The reaction was analyzed by HPLC and showed complete conversion of 2 (31 min) to the desired product 3 (16 min) as shown in Figure 2. Method B. To 0.127 mL of a 0.79 mM solution of ODN 1 (0.10 µmol) were added 0.1 mL of 1 M sodium borate buffer (pH 8.3) and 0.3 mL of 0.1 M sodium borate buffer
Bioconjugate Chem., Vol. 8, No. 2, 1997 239
(pH 8.3) in a 1.7 mL Eppendorf tube. A solution of 4-iodobenzoyl chloride (2.5 mg, 9.4 µmol) in 0.5 mL of THF was added, and the solution was kept at room temperature for 16 h. HPLC showed complete reaction of 1 (8.7 min). The reaction mixture was diluted to 2 mL and purified by centrifugal ultrafiltration through a 1000 MW cutoff concentrator (Filtron). This process removed excess hydrolyzed acid chloride and gave the desired product 3 in 97% purity as evidenced by HPLC. The retentate was diluted to 0.5 mL with water, and concentration was determined by A260 measurement. Recovery of 3 was 0.41 mg (77% yield). This product coeluted with 3 prepared according to method A and was used for thermal denaturation experiments. Thermal Denaturation Studies. The hybridization properties of all modified ODNs were examined by forming duplexes with complementary ODN 4 and determining the melting temperatures (Tm). Each ODN was present at 2 µM in pH 7.2 PBS (9.2 mM disodium phosphate, 0.8 mM monosodium phosphate, 0.131 M sodium chloride). UV absorbance was measured as the samples were heated from 10 to 90 °C with a temperature increase of 0.5 °C/min. Thermal dissociation curves were obtained from A260 vs temperature. The Tm was determined from the derivative maximum. Data from one representative run are given in Table 1. Radiolabeling Procedures. The volatile nature of 125I dictates certain handling precautions. Radiohalogenation of ODNs was carried out in a Plexiglas “iodination box”, which had its own exhaust fan and charcoal filter (Radiation Physics, Beltsville, MD) and which was placed within a fume hood. 125I was handled using standard syringe techniques. Labeling reactions were conducted in a reaction vessel that was vented to the inside of the iodination box through a 10 mL syringe filled with charcoal. Double gloves were routinely used to prevent the volatile halogens from penetrating the first glove layer. Commercially available “sleeve protectors” were used to decrease contamination of lab coats. Monitoring with a Geiger counter was conducted continually throughout an experiment. Between 24 and 72 h postlabeling, investigators should have a thyroid bioassay conducted. Conversion of 2 to [125I]ODN (3). To a solution of 11 µg (2 nmol) of Sn-ODN 2 in 22 µL of 0.1 M borate buffer (pH 8.3) were added 1.41 mCi (∼0.64 nmol, 2200 Ci/mmol) of Na125I in 3 µL of 0.1 M NaOH (New England Nuclear) and 20 µg (88 nmol) of ChT in 20 µL of water. After 15 min, the reaction was quenched with 10 µL of sodium bisulfite (10 mg/mL). A 5 µL aliquot of the crude reaction mixture was analyzed by radio-HPLC. The γ-detector indicated 69% conversion of 125I to the desired I-ODN 3 (22 min peak) as shown in Figure 3. The remaining [125I]ODN reaction mixture was loaded on a Poly-Pak reversed phase syringe cartridge (Glen Research) and eluted first with 2 mL of TEAA and then with 2 mL of 20% acetonitrile in TEAA. Fractions of ∼0.5 mL were collected, and each was measured in the dose calibrator. The most concentrated fraction (no. 5) contained 0.384 mCi (27% radiochemical yield) of [125I]ODN 3. Radio-HPLC analysis of this sample showed 96% purity. Earlier radioactive fractions were not analyzed for purity. Alternatively, the [125I]ODN product was isolated from the reaction mixture by reversed phase HPLC. In a separate labeling run, 50 pmol of 2 in 20 µL of 50 mM borate buffer (pH 8.5) was treated with 350 µCi (∼160 pmol) of Na125I and 20 µg of ChT. After 10 min, the reaction was quenched with 10 µL of sodium bisulfite (10 mg/mL). The mixture was passed through a Sephadex G-50 microspin column (Pharmacia). The product (3) was
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Figure 1. Synthesis of I-ODN conjugates. Reagents: (a) 4-(trin-butylstannyl)-benzoate, NHS ester; (b) Na*I, chloramine T; (c) 4-iodobenzoyl chloride.
recovered in 50 µL of TES buffer buffer (50 mM Tris, pH 8, 50 mM NaCl, 1 mM EDTA) and contained 25 µCi of 125 I while 320 µCi was retained by the column. HPLC purification used a 150 × 4.6 mm C18 column (Supelcosil LC-18-T) and the gradient specified in Figure 2. The radioactive peak corresponding to product (11-16 min) was collected in 0.5 mL fractions. Fractions 3-5 (3.8 µCi, 1.73 pmol) were combined and taken to dryness on the Speed Vac. The residue was dissolved in 10 µL of TES buffer. [125I]ODN 3 appeared as a single band by both denaturing and nondenaturing PAGE. Duplex Preparation with [125I]ODN (3). The 42mer target (ODN 4) complementary to ODN 3 was purified by gel electrophoresis prior to 5′-32P labeling with [32P]-γ-ATP using T4 polynucleotide kinase. The product was purified on a Pharmacia Microspin G50 gel filtration column according to the manufacturer’s protocol, and its final concentration was estimated to be 0.25 pmol/µL on the basis of quantitative recovery of ODN. HPLCpurified [125I]ODN 3a (0.4 pmol, 2.5 µL) and ODN 4 (0.25 pmol, 1 µL) were annealed at 40 °C for 10 min in 1× TES buffer. Two different duplex solutions were prepared with final volumes of either 25 µL (sample D1) or 10 µL (sample D2). The products of annealing were analyzed in 20% native PAGE as shown in Figure 4. Extent of duplex formation was quantitated using a FUJI BioImager BAS1500 and MacBAS software. DNA Strand Break Analysis. After 15 days at -70 °C, the duplex samples D1 and D2 described above were analyzed for fragmentation of the ssDNA target by 10% denaturing PAGE as shown in Figure 5 (panel A). The bands corresponding to fragmentation of each duplex were quantitated on the FUJI BioImager, and the distribution of breaks in [32P]ODN 4 for sample D2 are presented in Figure 5 (panel B). A control sample containing [32P]ODN 4 but no [125I]ODN 3a showed trace bands that were subtracted as background. RESULTS
The conjugation and iodination chemistry were developed using the nonradioactive isotope of iodine (127I). As shown in Figure 1, two methods were used to prepare iodinated ODNs, but only method A was used to prepare 125I-labeled ODNs. An amine-modified ODN (1) was treated with N-hydroxysuccinimidyl p-tributylstannyl-
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Figure 2. HPLC chromatograms describing synthesis of I-ODN 3 from Sn-ODN 2, as shown in Figure 1 (method A). The HPLC system used a 250 × 4.6 mm C18 column (Rainin Dynamax 300 Å) and a gradient of 5-85% solvent B over 40 min (flow rate ) 1 mL/min), where solvent A ) 0.1 M triethylammonium acetate (pH 7.5), solvent B ) acetonitrile; detection was by UV absorbance at 260 nm. Chromatogram for ODN 3 shows the reaction mixture 5 min after treatment of ODN 2 with NaI and chloramine T.
benzoate to give the key Sn-ODN precursor (2). Conversion to the desired I-ODN (3) was accomplished by treatment with excess NaI and ChT. To confirm the structure of the I-ODN prepared from Sn-ODN intermediate, I-ODN (3) was also prepared directly from 1 by reaction with 4-iodobenzoyl chloride (method B). The structure and sequence of the starting aminemodified ODN (1) are shown in Figure 1. The ODN sequence is a 15-mer that is complementary to the initiation codon region of the c-myc oncogene. The 3′hexanol modification was introduced into the ODN to improve serum stability (10). The 5′-hexylamine linker group was readily introduced into 1 on the DNA synthesizer using a commercially available phosphoramidite. Preparation of the Sn-ODN was complicated by poor water solubility of the lipophilic tributyltin NHS ester. Suitable reaction conditions for synthesis of 2 used an excess of the NHS ester in an emulsion of THF and borate buffer (pH 8.3). Reversed phase HPLC analysis showed complete conversion of 1 to 2 after several hours. As shown in Figure 2, the lipophilic Sn-ODN (31 min retention time) was isolated by HPLC in high purity. Only 42% yield was obtained, presumably due to losses in the prior filtration step. The organometallic product destannylates under acidic conditions, so the Sn-ODN solutions were stored at pH 8 or above. These solutions had good stability (90% recovery and our prior experience with these columns. Two different duplex solutions were prepared with different ODN 3 concentrations. These preparations were assayed for duplex formation by nondenaturing PAGE as shown in Figure 4. The less concentrated sample (D1) showed 65% of the slower moving duplex band, whereas sample D2 showed 89% duplex formation. As expected, both samples showed unhybridized I-ODN. The samples were frozen in liquid nitrogen and kept at -70 °C for strand break analysis of ODN 4. After 15 days of storage, the [125I]ODN 3a/[32P]ODN 4 duplexes were analyzed for fragmentation by denaturing PAGE as shown in Figure 5A. The length of the 5′-32Plabeled fragments in lanes 1 and 2 provides evidence for sequence specific cleavage by 125I decay. The position of the breaks relative to the sequence of ODN 4 was determined by counting the bands from the top of the gel. Breaks at a given position are due to complete removal of the corresponding base and result in a fragment n - 1 nucleotides long, where n is the position
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Figure 5. (A, top) 10% denaturing PAGE showing fragmentation of ODN 4 after 15 days of decay. Lanes: (1) duplex D1; (2) duplex D2; (3) [32P]ODN 4; (4) [125I]ODN 3. (B, bottom) Bar graph showing distribution and relative intensity of breaks at individual bases of ODN 4 for duplex D2. X indicates position of attachment of iodobenzamide conjugate group. Total yield of strand breaks was 4.6% for D1 and 6.6% for D2.
of the nucleotide from the 5′-end. For example, a break at T34 (maximum in Figure 5B) corresponds to the 33 nucleotide long fragment in the gel. The bar graph in Figure 5B shows the relative intensity of these cleavage bands for the more concentrated duplex sample (D2) and how their position corresponds to the sequence of ODN 4. The pattern of breaks for sample D1 was identical, but the intensity of each band was slightly lower. The value of each bar represents percent intensity of the band compared to the sum of the intensities of all bands including the top band (unbroken ODN 4). The total yields of the strand breaks were calculated to be 4.6% for sample D1 and 6.6% for sample D2. DISCUSSION
Before the advent of automated ODN synthesis, 125Ilabeled DNA probes were obtained by direct treatment with thallium chloride and sodium iodide at elevated temperature (14). The oxidized form of 125I (I+) reacts with random cytosine bases in the DNA to give 5,6saturated pyrimidine residues. The harsh reaction conditions degraded the DNA and gave mixtures of iodinated products. Direct iodination of the cytosine residues is undesirable since the modified bases compromise hybridization properties of shorter probes. Automated synthesis of 3′-labeled [125I]ODNs from a [125I]dU-modified solid support has been reported (15). The I-ODN products were used as PCR primers for preparation of internally modified [125I]ODNs. This method is unsuitable for labeling with shorter half-life radiohalogens. More direct methods for radioiodination
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of ODNs use conjugate groups that can be selectively iodinated. For example, 5′-hexylamine-modified ODNs have been chemically conjugated to 4-hydroxyphenyl (16) or 4-methoxyphenyl groups (17). Recently, a phosphoramidite reagent has been developed that allows direct incorporation of 4-hydroxyphenyl groups into 5-methyl dC nucleotides using a DNA synthesizer (18). To ensure that no cytosine residues react (and to speed the reaction), the modified ODN is generally present in large excess with respect to the small quantities of radioiodine that are used. Unreacted (carrier) ODN must be separated from the I-ODN since it competes for complementary nucleic acid target sites. This separation is difficult for the conjugates studied to date. The radioiodination method reported here uses a tributylstannylbenzamide precursor (Sn-ODN 2). The major advantage of this new radiolabeling procedure is that unreacted Sn-ODN and free iodine are easily separated from the desired I-ODN product. The lipophilic organometallic precursor was soluble and stable in aqueous buffer (pH 8.3). The hexylamine linker arm in ODN 1 reacted cleanly with tributylstannylbenzoic acid NHS ester to give 2. This active ester has been used for labeling monoclonal antibodies with a number of radiohalogens (19). The strong UV absorbance of ODNs allowed the iodination chemistry to be developed using 127 I and reversed phase HPLC methods. Conversion of the Sn-ODN to I-ODN 3 proceeded quantitatively when excess NaI was used. The identity of 3 was confirmed by preparation via a more direct route (Figure 1, method B). Tm studies showed that the addition of the 4-iodobenzamide conjugate group did not adversely affect hybridization. Radioiodination of ODN 2 with 125I gave 69% conversion to [125I]ODN 3. The large shift in retention time allowed easy isolation and identification of the I-ODN product. Reversed phase chromatography using a disposable syringe cartridge gave 27% isolated yield of no carrier added [125I]ODN 3 in 96% purity. A large excess of Sn-ODN was not required to drive the reaction, but a slight excess ensured complete reaction of the relatively expensive radioisotope. Although a C18 stationary phase was useful for analytical HPLC, recovery was poor in a preparative mode. We are experimenting with other reversed phase packings and HPLC methods to improve radiochemical yields. The hybridization and DNA cleavage properties of [125I]ODN 3 were studied using gel electrophoresis assays. We were especially interested in the effect of the relatively long 5′-linker in 3 on DNA cleavage efficiency. The reported 15-20 Å cleavage range (1) of [125I]dC or [125I]dU-modified ODNs hybridized to ssDNA targets may be an ideal situation. Substitution of 125I at the 5-position of the pyrimidine ring essentially “locks” it into the major groove, approximately 5 Å from the axis of the B-form DNA helix. We suspected that the hexylaminebenzamide linker arm used to attach 125I to the 5′-terminus might be too long (∼16 Å) and flexible to allow efficient DNA cleavage. Nondenaturing PAGE analysis of the duplex formed between [125I]ODN 3 and 42-mer ODN 4 showed that extent of duplex formation was concentration dependent. Duplex sample D1 (10 nM in 4) showed 65% dsDNA, whereas sample D2 (25 nM in 4) showed 89% dsDNA. After 15 days (16% 125I decay), the duplex samples were assayed for strand breaks using denaturing PAGE (Figure 5). Samples showed a Gaussian distribution of DNA fragments typical for strand breaks caused by 125I. Taking into account that only 65% and 89% of ODN 4 formed duplexes, we recalculated the effective yield of
125I-Labeled
Bioconjugate Chem., Vol. 8, No. 2, 1997 243
Oligonucleotides
breaks as 7.1% and 7.4% for D1 and D2, correspondingly. Therefore, the yields in terms of breaks per decay were 0.44 and 0.46. It is interesting that the DNA fragment of maximum intensity corresponds to the position of attachment of the 5′-terminal [125I]benzamide containing linker. If this linker were stretched out in either the 5′- or 3′-direction, then the pattern of bands would be expected to shift (up to five nucleotides). The high cleavage efficiency (0.45 break per decay) indicates that the 125I atom is constrained close to the sugar-phosphate backbone of the target DNA strand, much like the geometry obtained from labeling at the 5-position of the pyrimidine ring. It is not clear how the DNA cleavage efficiency of these different linker systems compares since no values have been reported for cleavage of ssDNA targets with 125Ilabeled dC or dU. Results showed 0.8 ds break per decay from 125I-dC-labeled triplex forming ODNs that lie in the major groove of target dsDNA (3). Further investigations are planned to explore the effect of different linker systems on the DNA cleavage properties of 125I. For example, 5-(aminoalkyl)uridine linkers can be used to prepare internally modified ODNs that constrain the iodobenzamide group in the major groove of duplex DNA. The rapid radioiodination method reported here should also allow the DNA cleavage properties of 123I to be explored. LITERATURE CITED (1) Martin, R. F., and Haseltine, W. A. (1981) Range of radiochemical damage to DNA with decay of iodine-125. Science 213, 896. (2) Charlton, D. E., and Booz, J. (1981) A Monte Carlo treatment of the decay of 125I. Radiat. Res. 87, 10. (3) Panyutin, I. G., and Neumann, R. D. (1994) Sequencespecific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides. Nucleic Acids Res. 22, 4979. (4) Bloomer, W. D., and Adelstein, S. J. (1977) 5-125I-iododeoxyuridine as prototype for radionuclide therapy with Auger emitters. Nature 265, 620. (5) Kassis, A. I., Adelstein, J., and Bloomer, W. D. (1987) Therapeutic Implications of Auger-Emitting Radionuclides. Radionuclides in Therapy (R. P. Spencer et al., Eds.) pp 120134, CRC Press, Boca Raton, FL. (6) Makrigiorgos, G. M., Kassis, A. I., Baranowska-Kortylewisz, J., McElvany, K. D., Welch, M. J., Sastry, K. S. R., and Adelstein, S. J. (1989) Radiotoxicity of 5-[123I]iodo-2′-deoxy-
uridine in V79 cells: a comparison with 5-[125I]iodo-2′deoxyuridine. Radiat. Res. 118, 532. (7) DeSombre, E. R., Shafii, B., Hanson, R. N., Kuivanen, P. C., and Hughes, A. (1992) Estrogen receptor-directed radiotoxicity with Auger electrons: specificity and mean lethal dose. Cancer Res. 52, 5752. (8) Makrigiorgos, G. M., Berman, R. M., Baranowska-Kortylewicz, J., Bump, E., Humm, J. L., Adelstein, S. J., and Kassis, A. I. (1992) DNA damage produced in V79 cells by DNA-incorporated Iodine-123: a comparison with iodine-125. Radiat. Res. 129, 309. (9) Coenen, H. H., Moerlein, S. M., and Stocklin, G. (1983) Nocarrier-added radiohalogenation methods with heavy halogens. Radiochim. Acta 34, 47. (10) Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A. A., Scholler, J. K., and Meyer, R. B. (1993) Facile preparation of nuclease resistant 3'-modified oligodeoxynucleotides. Nucleic Acids Res. 21, 145. (11) Reed, M. W., Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr. (1991) Acridine and cholesterol derivatized solid supports for improved synthesis of 3′-modified oligonucleotides. Bioconjugate Chem. 2, 217. (12) Cantor, C. R., Warshaw, M. M., and Shapiro, H. (1970) Oligonuclotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers 9, 1059. (13) Wilbur, D. S., Hadley, S. W., Hylarides, M. D., Abrams, P. G., Beaumier, P. L., Morgan, A. C., Reno, J., and Fritzberg, A. R. (1989) Development of a stable radioiodinating reagent to label monoclonal antibodies for radiotherapy of cancer. J. Nucl. Med. 30, 216. (14) Commerford, S. L. (1971) Iodination of nucleic acids in vitro. Biochemistry 10, 1993. (15) Scherberg, N., Bloch, I., and Gardner, P. (1992) Site-specific incorporation of [125I]iododeoxyuridine into DNA. Appl. Radiat. Isot. 43, 923. (16) Dattagupta, N., and Knowles, W. EP 0198207. (17) Dewanjee, M. K., Ghafouripour, A. K., Werner, R. K., Serafini, A. N., and Sfakianakis, G. N. (1991) Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique. Bioconjugate Chem. 2, 195. (18) Fontanel, M. L., Bazin, H., Roget, A., and Te´oule, R. (1993) Synthesis and use of 4-hydroxyphenyl derivatized phosphoramidites in the selective radioiodination of oligonucleotide probes. J. Labelled Compd. Radiopharm. 33, 717. (19) Wilbur, D. S. (1992) Radiohalogenation of proteins: an overview of radionuclides, labeling methods, and reagents for conjugate labeling. Bioconjugate Chem. 3, 433.
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