Bioconjugafe Chem. 1995, 6, 174-1 78
174
Versatile Procedure of Multiple Introduction of 8-Aminomethylene Blue into Oligonucleotides Uwe Moller,” Frank Schubert, a n d Dieter Cech Humboldt-Universitat zu Berlin, Institut fur Chemie, Hessische Strasse 1-2, 10099 Berlin, Germany. Received August 8, 1994@
The coupling of 8-aminomethylene blue to oligonucleotides via poly-L-glutamic acid linker using carboxy-anchor groups will be described. The introduction of carboxy-anchor groups into oligonucleotides proceeds both during automated synthesis using 6-(ethoxycarbonyl)hexyll-O-phosphoramidite and by reaction of 5’-amino-functionalized oligonucleotides with succinic anhydride. 0-(N-Succinimidyl)-l,1,3,3-tetramethyluronium tetrafluoroborate was used as activating reagent for binding of poly- L-glutamic acid to the carboxylated oligonucleotides. The successful 5’-carboxylation and polyL-glutamic acid coupling were proven both by polyacrylamide gel electrophoreses and HPLC. 8-Aminomethylene blue in its leucoform was covalently coupled to the oligonucleotides in the presence of water soluble carbodiimide.
INTRODUCTION
Various dye conjugates with proteins, nucleic acids, and carbohydrates have been synthesized and are currently being studied for their analytical, biochemical, and medical application. The covalent attachment of dyes to biomolecules has proven to be advantageous in many approaches. Thus, different strategies of introduction of photoactive molecules onto functional groups have been developed. Dye-biomolecule conjugates now have a variety of applications from fluorescent detection of DNA fragments in semiautomated sequencing (Smith et al., 19871, to the fluorescent immuno assays (FIA), and in addition, to the investigation of the biochemical mechanism of membrane protein interaction (Garland and Moore, 1979). More recently, however, these conjugates have developed to such an extent that they are also being used as therapeutic agents. Thus, photosensitizers which are able to generate singlet oxygen photochemically are frequently found in clinical trials of tumors (the so-called photodynamic therapy of tumors; Dougherty et al., 1987). Furthermore, site-directed DNA damaging using oligonucletides tethered to dyes stimulated by photochemical formation of singlet oxygen has been described (Buchardt et al., 1989; Fedorova et al., 1990). It was shown in clinical trials of tumors by PDT that the specificity was increased by introduction of photosensitizers into antibodies, which are complementary to targeted surface antigenes of tumors. For that, the utility of labeled antibodies could be considerably enhanced if the label is multiply conjugated to the biopolymer. In this context, polyvinyl alcohol and polyamines served as anchor groups due to their ability to couple more than one dye molecule in a definite manner (Jiang et al., 1990). 3,7-(Dimethy1amino)phenazathioniumchloride (methylene blue) was shown t o be a very effective singlet oxygen sensitizer in organic photochemistry. Its photochemical properties and noncovalent interactions with nucleic acids and proteins were intensivly investigated (Tuite and Kelly, 1993). Therefore, the covalent introduction of methylene blue into various biomolecules and
* Author to whom correspondence should be addressed. Abstract published in Advance ACS Abstracts, January 1, 1995. @
the study of the photochemical behavior of such conjugates was of particular interest. Our recent studies of methods to incorporate different dyes into oligonucleotides for a variety of applications encouraged us to synthesize oligonucleotide-methylene blue conjugates with more than one dye molecule within the oligonucleotide (Moller et al., 1990; Schubert et al., 1990, 1994). Conceptually, poly-L-glutamic acid and methylene blue in any derivatized form served as starting compounds. Commercial available poly-L-glutamic acid has the advantage that its single N-terminal amino group favors the attachment to carboxy-alkylated oligonucleotides and was synthesized as described in the literature (Kremsky et al., 1987). The principle obstacle to the covalent coupling of methylene blue rests in the lack of any functionality within the molecule. Therefore, 8-aminomethylene blue which is easily available from methylene green seems to be a potential candidate for covalent coupling to the polylinker. The present report describes the synthesis of oligonucleotides with a poly-L-glutamic acid linker and demonstrates that the linkage to the methylene blue derivative can be stably formed. EXPERIMENTAL PROCEDURES
All reagents used were reagent grade or better. PolyL-glutamic acid was purchased from Sigma, TSTU from Calbiochem-Novabiochem (Bad SodedGermany), and methylene green from Aldrich. Oligonucleotides were synthesized on a Gene Assembler Plus DNA Synthesizer (Pharmacia LKB) by a n automated phosphoramidite method (commercially available phosphoramidites with benzoyl and isobutyryl as base protecting groups on G, A and C were used). 5’-Aminofunctionalization was carried out with 6-((trifluoroacetyl)amino)hexyl-2-cyanoethyl NJV-diisopropylphosphoramidite(Pharmacia LKB). ‘H- and 31P-NMRand absorption spectra were recorded on a Bruker AM 300 spectrometer and on a Shimadzu U V 160 spectrophotometer, respectively. Analytical HPLC measurements were performed on an ICUGAT system using a LiChrosorb RP18 column (125 x 4 mm, 5 mm) and an acetonitrile containing triethylammonium acetate buffer gradient (buffer A: 50% 0.1 M TEAAC pH 6.5, 50% CH&N; buffer B: 98% 0.1 M TEAAC pH 6 . 5 , 2% CH3CN; gradient: linear from 90% buffer B to 40% buffer B in 40 min; flow rate: 1 mL/ min) as eluent. Preparative HPLC was carried out on a
1043-1802/95/2906-0174$09.00/0 0 1995 American Chemical Society
Bioconjugafe Chem., Vol. 6,No. 2, 1995 175
8-AminomethyleneBlue-Oligonucleotide Conjugates Hyperprep RP18 column (125 x 16 mm, 12 mm; GAT, BremerhavedGermany) with a flow rate of 5 m u m i n using the same system and gradient.
Synthesis of 5'-Carboxy-Modified Oligonucleotides 2. Path A. The phosphoramidite of commercially available ethyl 6-hydroxyhexanoate was synthesized according to standard protocols (McBride and Caruthers, 1983) with 2-cyanoethyl Nfl-diisopropylchlorophosphoramidite in 91% yield. The phosphoramidite building block was coupled to the 5'-end of oligonucleotides during automated syntheses using standard protocols. After treatment with 0.1 M sodium hydroxide (24 h a t 40 "C) and purification by HPLC on reversed-phase (Hyperprep RP 18) the carboxymodified oligonucleotides 2a were obtained in good yields and in high purity. The coupling yield of the 6-(ethoxycarbony1)hexyl 1-0-phosphoramidite (6-ECHP)' estimated by integration of HPLC chromatograms was approximately 90%. Path B. For carboxylation, 100 nmol of amino-functionalized oligonucleotide was dissolved in 50 p L of water and mixed with 50 pL of an aqueous solution of SA (50 mg/mL). After the mixture was shaken for a period of 1 h further, 50 pL of the SA solution was added. After a n additional hour the modified oligonucleotide was purified on Sephadex G-25 (NAP-10, Pharmacia LKB). The obtained oligonucleotide was evaporated and then treated with 500 pL of concentrated ammonia for 0.5 h a t room temperature. Chromatography on Sephadex G-25 and final purification by HPLC yielded the oligonucleotide 2b (X = -(CH&NH(CH2)2-). Preparation of Poly-L-glutamicAcid Oligonucleotide Conjugates 5. The carboxy-modified oligonucleotide 2 (10 nmol) was dissolved in 2 pL of water and then diluted with 20 pL of dimethylformamide (free of amines). O-(N-Succinimidyl)-l,1,3,3-tetramethyluronium tetrafluoroborate (9 pL, 30 nmol) dissolved in dimethylformamide (10 pg/lO pL) and diisopropylethylamine(10 mL, 30 nmol) in dimethylformamide ( 5 x lT3pU10 pL) were added. The mixture was stirred for 1h, and without any further purification 50 pL (100 nmol) of a n aqueous solution of P-L-G~u (19.4 mg/mL) and 8.6 pL (100 nmol) of diisopropylethylamine diluted with dimethylformamide (20 x pU10 pL) were added. After 24 h the reaction mixture was evaporated to dryness in vacuo. The isolation of conjugates was carried out by polyacrylamide gel electrophoreses (PAGE). Synthesis of the Leucoform of 8-Aminomethylene Blue 7. Commercially available methylene green (6)was purified by silica gel chromatography using acetonitrile: water (5050 = v:v, 0.1 M NaC1). The purified methylene green (18 mg, 50 pmol, calculated to 1pmol of carboxy groups) was dissolved in 2.5 mL of a mixture of dioxane:water (2:l = v:v) and mixed with palladiudcharcoal. The dye was reduced in a hydrogen atmosphere under normal pressure. After the color of methylene green disappeared, the mixture was stirred for another 0.5 h. Without any isolation of the leucoform, the obtained solution was used for coupling experiments after removal of the catalyst under exclusion of oxygen. Conjugation of 8-AminomethyleneBlue to PolyL-glutamicAcid Modified Oligonucleotides. Imida-
Chart 1. 8-Aminomethylene Blue p-L-Glu-Oligonucleotide Conjugate. 1
I-
m
X = -(CH,),-NH-CO-(CH2)2- ; -(CH2),. n = 12.30 : m = -60
zole (68 mg, 1 mmol) was dissolved in 2.4 mL of MES buffer (0.1 MI, and 100 pL of an aqueous solution of a p-L-Glu-oligonucleotideconjugate (1pmol, compared to carboxyl groups) as well as 15 mg (75 pmol) of N43(dimethy1amino)propyl)-N'-ethylcarbodiimide hydrochloride were added. The solution was gassed with nitrogen for 5 min, and the leucoform of 8-AMB obtained above was added under exclusion of oxygen. The coupling mixture was stirred in the dark a t room temperature for 24 h. In order to oxidize the dye, the reaction mixture was gassed with oxygen for 10 min. The dark blue solution was evaporated to dryness in vacuo. Conjugate 1 was isolated by dialysis against PBS. RESULTS
Labelling of p-L-Glu-OligonucleotideConjugates with 8-AMB. Commercially available methylene green has served as starting material for labeling reactions because it can easily be reduced to 8-AMB. However, the commercial form contains only 60% of methylene green, and a liquid chromatographic purification on silica gel needs to be done before use. Then the purified methylene green was reduced by palladiudcharcoal in a hydrogen atmosphere to the leucoform of 8-AMB. The formation of the leucoform can easily be pursued on the decolorization of the reaction mixture. Finally, after successful coupling to the oligonucleotide the leucoform was converted into the stable oxidized form by oxygen. However, the reaction of 8-AMB with poly-L-Glu-oligonucleotide conjugates did not result in the wanted conjugate 1 (Chart 1). Since 8-AMB in its oxidized form is positively charged in the aromatic system the exocyclic amino group in position 8 does not have a sufficient nucleophilicity for a reaction with carboxyl groups. Therefore, it seems to be necessary to transform the methylene blue derivative in its leucoform. For that, the described synthesis of 8-AMB was stopped a t the level of its leucoform by subsequent exclusion of oxygen. The following coupling with the carboxyl groups of p-L-Glu-oligonucleotide conjugates 5 (Scheme 1)was carried out in a mixture of buffer (MEW and dioxane under a n inert atmosphere in the presence of water soluble carbodiimide. After 24 h the reaction mixture was gassed shortly with oxygen to get back 8-AMB by a complete oxidation of the leucoform. The Abbreviations used: P-L-G~u, poly-L-glutamic acid; 8-AMB, conjugates were separated from unreacted dye by dialysis 8-aminomethylene blue; PAGE, polyacrylamide gel electrophoresis; TSTU, O-(N-succinimidyl)-l,1,3,3-Btramethyluronium against PBS. Additional purification by chromatography on Sephadex G25 and on silica gel Si60 did not show tetrafluoroborate; DMF, dimethylformamide; SA, succinic anunlabeled p-L-Glu-oligonucleotide conjugates in the hydride; 6-ECHP, 6-(ethoxycarbonyl)hexyl 1-0-phosphoramiddialyzed product. The isolated dye conjugates display a ite; MES, 2-morpholinoethanesulfonic acid; PBS, phosphatebuffered saline; TEAAC, triethylammonium acetate. typical absorption maximum for oligonucleotides a t 260
M6ller et al.
176 BioconjugafeChem., Vol. 6, No. 2, 1995
Scheme 1. Labeling of p-cGlu-Oligonucleotide Conjugates with the Leuco Form of 8-AMB.
1
2 0.5 m
n
0 0
5
10
15
20
25
30
20
25
30
t In mln
1.
EDAC
6
pH6
Pd I H2
1
nm, and compared to free 8-AMB, the &-band of conjugates has been shifted hypsochromically at 15 nm. Synthesis of 5’-Carboxy-Modified Oligonucleotides. In order to incorporate numerous carboxyl anchor groups in oligonucleotides for labeling with 8-kVB it was advantageous to use P-L-G~u.Thus, 5’carboxy prefunctionalized oligonucleotides were suitable for its introduction. In the present work two routes of introduction of a carboxy group at the 5’-end of oligonucleotides were studied. In path A 6-(ethoxycarbonyl)hexyl 1-0-phosphoramidite (6-ECHP) is used as a building block in automated oligonucleotide synthesis via the phosphoramidite approach. According to standard protocols (McBride and Caruthers, 1983; Kremsky et al., 1987) the synthesis of the phosphoramidite was performed using ethyl 6-hydroxyhexanoate and 2-cyanoethyl NJV-diisopropylchlorophosphoramidite. 6-ECHP was obtained in 91% yield. and IH-NMR indicated the high purity of the product both after distillative and aqueous isolation (data not shown). Carboxyphosphoramidite dissolved in acetonitrile was used in the last cycle of oligonucleotide synthesis under standard conditions. In contrast to the usual deblocking of protecting groups it is recommended to use 0.1 M sodium hydroxide (24 h, 40 “C) for complete deblocking of the exocyclic amino groups as well as the ethyl ester of the carboxy linker. After purification by reversed-phase HPLC 5’-carboxymodified oligonucleotides 2 (X = -(CH&-) were obtained in a yield varying from 20% to 58% due to the reaction conditions. An enzymatical digestion has shown that no base desamination was obtained. Chromatogram I in Figure 1shows a preparative HPLC profile of oligonucleotide 2a. The very simple reaction of amines with succinic anhydride (SA) is shown in path B. First, 5’-amino functionalization was performed with commercially available phosphoramidites. The fully unprotected oligonucleotide was then reacted with SA in water for 2 h. After the excess succinic anhydride was removed by Sephadex G25 chromatography the sample was treated with concentrated ammonia (30 min, rt)to cleave succinic ester at the 3’-end of oligonucleotides leaving the 5’-
0
5
10
15
t In mln Figure 1. HPLC chromatograms of 3’-TGA CCG GCA GCA AAA TGT TGC AGC(CH2)&OOH-5’ (I, preparative run of the crude product) and of a 1:l-mixture of the same oligonucleotide and its unmodified analogue (11). A: unmodified oligonucleotide. B: 5’-carboxy-modified oligonucleotide. See the Experimental Procedures for detailed HPLC conditions.
1
2
3
4
Figure 2. 20%PAGE of B’-carboxylated oligonucleotides and their starting sequences. Lanes: (1) 3’-TGA CCG GCA GCA AAA TGT TGC AGC-5’, (2) 3’-TGA CCG GCA GCA AAA TGT TGC AGC(CH2)&OOH-5’, (3) 3’-(T)*0(CH2)aNH2-5’,(4) 3’-(T)20
(CH2)3NHCO(CH2)2COOH-5’.
terminus uneffected. A final purification on Sephadex G25 resulted in 2b with X = -(CH&NH(CH&-. In order to estimate whether carboxyl groups are coupled at the %-end of oligonucleotides, polyacrylamide gel elctrophoresis (PAGE) was performed. The succinylation has only caused a slight decrease in the electrophoretically mobility. Lanes 3 and 4 in Figure 2 show the small difference between a succinylated 5’-amino oligonucleotide and its unmodified sequence. p-cGlu- Oligonucleotide Conjugates. A fraction of the polydisperse amino acid having a molecular weight of 10 000 was chosen for coupling of p-L-Glu (3)with the 5’-carboxy modified oligonucleotides 2. This implicates more than 60 anchor groups per oligonucleotide. In this case the coupling strategy of activated esters (Scheme
8-Aminomethylene Blue-Oligonucleotide Conjugates
Sioconjugate Chem., Vol. 6, No. 2, 1995 177
Scheme 2. Coupling of p-~-Glu to B’-CarboxyFunctionalized Oligonucleotides. El
‘‘
.
2 -
-.
0
o-oJ,
1
.-
II
0-P-0-X-COOH
Ho+
n
2a, b
e BF.
COOH
I
conjugate. Lanes: ( 1 ) ~’-(T)~O(CH~~NHCO(CH~)~COOH-~’, (2)
y42
y
U Figure 3. 20%PAGE of an unpurified p-LGlu-oligonucleotide
~‘-(T)~~(CH~)BNHCO(CH~)~CONH-~-I,-G~U-~’.
2
yields. In analogy to the synthesis of nucleoside phosphoramidites 6-ECHP can be obtained in yields of about m 95%. With use of 6-ECHP the oligonucleotide is modified 4 at the end of the automated synthesis, which in contrast to the postsynthetic procedure is not time-consuming and does not need additional chromatographic purifications. Furthermore, in order to dissolve the phosphoramidite of the carboxylinker during automated oligonnucleotide synthesis, it is not necessary to add pyridine as it is 0 essential for long chain derivatives (Kremsky et al., II 1987). The coupling of the modified phopshoramidites is very eficient giving pure products as shown in the HPLC-chromatogram I in Figure 1. The HPLC profile n shows a small background of side products (profile I), and Sa, b a separation of the modified (B) from the unmodified sequence (A)on reversed phase HPLC is possible (profile a: X = -(CH2)5-; b: X = -(CH2),-NH-CO-(CH2),-11). The introduction of an alkyl chain into the oligonucleotide increases its hydrophobicity which causes a n = 12 - 30 ; m = -60 considerable increase in the retention time. Similarly, this effect also influences the mobility of oligonucleotide 2) was used. For that, O-(N-succinimidyl)-l,1,3,3-tet- sequences in polyacrylamide gel electrophoresis (lanes 1 ramethyluronium tetrafluoroborate (TSTU) served as a and 2 in Figure 2). coupling reagent. First, the Ei’-carboxyl group of modified Yields in path B using succinic acid are similar to that oligonucleotides was treated with TSTU (4) to form a n obtained using 6-ECHP. Contrary to path A, polyacrylactivated ester. Without further purification the actiamide gel electrophoresis is recommended for isolation vated oligonucleotides were added to an aqueous P-L-G~U and characterization of carboxylated oligonucleotides solution. Within 24 h the carboxylated oligonucleotides (lanes 3 and 4 in Figure 2). The slight structural reacted almost quantitatively to yield 5. In Figure 3 a differences between educts and products does not permit 20% PAGE of the crude product of a p-L-Glu-oligonuclea separation by HPLC. An advantage of the method otide conjugate is shown. As a result of higher molecular which proceeds in aqueous solution is given by its weight the mobility of the conjugate is decreased. possibility to carboxylate oligonucleotides at each position either at the 3’-or Ei’-end, at any modified nucleobases, DISCUSSION or at the phosphate backbone. For optimal generation of singlet oxygen an oligonucleFor the purpose of multiple coupling of 8-AMB to otide must be labeled as best as possible by a photosenoligonucleotides, it was decided to introduce P-L-G~u, sitizer. This requires attaching numerous photosensibecause the single amino group of P-L-G~Uallows a tizers like 8-AMB to each oligonucleotide. This should specific introduction of the 8-AMB to the carboxylated be achieved by a poly-L-glutamic acid polylinker which oligonucleotides. Thus, polydisperse P-L-G~Uwith a n is attached to the oligonucleotide by a carboxylic amide average of 60 monomer units was used. Longer polypepbond. Therefore, the oligonucleotide must be carboxytides were not used because there is a risk of precipitalated either during automated synthesis or postsynthetic tion. Although oligonucleotides are negatively charged after removal the oligonucleotide from the polymer to a certain extent, complex conjugates with aromatic support. Thus, for the synthesis of 8-AMB labeled substances tend to precipitate (Motsenbocker e t al., oligonucleotides two alternative routes for 5’-carboxy 1993). The synthesis of p-L-Glu-oligonucleotide conjumodification of oligonucleotides were studied. First, we gates 5 has proceeded in nearly quantitative yields with tested direct carboxylation during automated DNAall types of carboxy modified oligonucleotides. Lane 2 synthesis by 6-ECHP according to a described procedure in Figure 3 shows a gel electrophoresis of the crude by Kremsky et al. (1987).In the second method succinic product of such a coupling reaction. The slower running acid was used in the presence of water soluble carbodislight spot is due to the differences in molecular weigth imide giving satisfactory yields of the carboxylated oliof the used P-L-G~u. gonucleotide. Although both methods worked well, 5’All attempts to couple 8-AMB directly to the p-LGlucarboxylation by 6-ECHP is the most elegant way. This oligonucleotide conjugates failed due to the low nucleomethod is preferred as 6-ECHP is easily available in good philicity of the primary amino group. The nucleophilicity CH-COOH
Moller et al.
178 Bioconjugate Chem., Vol. 6,No. 2, 1995 ACKNOWLEDGMENT
I
This research was supported by the Bundesministerium fur Forschung und Technik of the Federal Republic of Germany (BE0/21-0310255A). LITERATURE CITED
400
500
600
700
800
nm
Figure 4. Absorption spectra of 8-AMB (I) and an 8-AMB-pL-Glu-oligonucleotide conjugate (11) at pH 7.
might be increased by formation of the leucoform reducing the electron-withdrawing effect of the heteroaromatic ring system. Indeed, the direct conjugation of 8-AMB in its leucoform was possible and proceeded in high yields. The high and different molecular weights did not allow analytical characterization of the obtained dye conjugates with conventional methods, as mass spectrometry and nuclear magnetic resonance until now. However, the comparison to authentic samples of 8-AMB and noncoupled p-L-Glu-oligonucleotide conjugates by thin layer chromatogaphy demonstrates that dye conjugates are not based on any ionic or intercalating interactions. In contrast to dye conjugates a mixture of both components was separated according to their characteristic Rfvalues. A further evidence for the covalent attachment of the dye is given by the UV spectra. The relatively small hypsochromic shift of the Sorret band of 8-AMB in the conjugate by 15 nm (curve I1 in Figure 4) does indicate formation of a covalent bond. These findings are in accordance with data found in the literature where ionic interactions of methylene blue derivatives with nucleic acids or proteins cause a blue shift of the Sorret band up to 70 nm and intercalating interaction with nucleobases a red shift of about 7 nm (Antony et al., 1993; Motsenbocker et al., 1993). Looking a t all the results together the described procedure demonstrates a n opportunity to simplify the coupling of a variety of dyes being less reactive under normal coupling conditions. In principal, dyes bearing thiol or hydroxy groups can be transformed to a higher state of nucleophilicity by the represented method. These include classes of dyes such as di- and triarylmethane dyes (i.e., derivatives of malachite green), acridines, phenoxazines, phenothiazines, and phenazines. Furthermore, the introduction of p-L-Glu as polylinker opens a way to attach numereous marker molecules to any biomolecule.
Antony, T., Atreyi, M., and Rao, M. V. R. (1993) Spectroscopic Studies on the Binding of Methylene Blue to Poly(riboadeny1ic acid). J . Biomol. Struct. Dyn. 11, 67-81. Buchardt, O., Karup, G., Egholm, M., Koch, T., Henriksen, U., Meldal, M., Jeppesen, C., and Nielsen, P. E. (1989) Photonucleases. Photochemical Probes i n Biochemistry (P. E. Nielsen, Ed.) 209-218. Dougherty, T. J. (1987) Photosensitizers: Therapy and Detection of Malignant Tumors. Photochem. Photobiol. 45, 879-889. Fedorova, 0. S., Savitskii, A. P., Shoikhet, K. G., and Ponomarev, G. V. (1990) Palladium(I1)-coproporphyrin I as a Photoactivable Group in Sequence Specific Modification of Nucleic Acids by Oligonucleotide Derivatives. FEBS Lett. 259, 335-337. Garland, P. B., and Moore, C. H. (1979) Phosphorescence of Protein-bound Eosin and Erythrosin. A Possible Probe for Measurements of Slow Rotational Mobility. Biochem. J . 183, 561-572. Jiang, F. N., Jiang, S.,Liu, D., Richter, A,, and Levy, J. G. (1990) Development of Technology for Linking Photosensitizers to a Model Monoclonal Antibody. J . Immunol. Methods 134, 139-149. Kremsky, J. N., Wooters, J. L., Dougherty, J. P., Meyers, R. E., Collins, M., and Brown, E. L. (1987) Immobilization of DNA via Oligonucleotides Containing an Aldehyde or Carboxylic Acid Group a t the 5’-Terminus. Nucleic Acids Res. 15,28912909. McBride, L. J., and Caruthers, M. H. (1983) An Investigation of Several Deoxynucleoside Phosphoramidites Useful for Synthesizing Oligodeoxyribonucleotides.Tetrahedron Lett. 24, 245-248. Moller, U., Cech, D., and Schubert, F. (1990) P(II1)-Acridine Derivatives as Building Blocks for the Solid-Phase Synthesis of Non-Radioactively Labelled Oligonucleotides. Liebigs Ann. Chem. 1221-1225. Motsenbocker, M., Masuya, H., Shimazu, H., Miyawaki, T., Ichimori, Y.,and Sugawara, T. (1993) Photoactive Methylene Blue Dye Derivatives Suitable for Coupling to Protein. Photochem. Photobiol. 58, 648-652. Schubert, F., Ahlert, K., Cech, D., and Rosenthal, A. (1990) OneStep Labelling of Oligo-nucleotides with Fluoresceine during Automated Synthesis. Nucleic Acids Res. 18, 3427. Schubert, F., Knaf, A., Moller, U., and Cech, D. (1994) Covalent Attachment of Methylene Blue to Oligonucleotides. Manuscript in preparation. Smith, L. M., Kaiser, R. J.,Sanders, J. Z., and Hood, L. E. (1987) The Syntheses and Use of Fluorescent Oligonucleotides in DNA Sequence Analysis. Methods Enzymol. 155, 260-300. Tuite, E. M., and Kelly, J. M. (1993) Photochemical Interactions of Methylene Blue and Analogues with DNA and other Biological Substrates. J. Photochem. Photobiol. B: Biol. 21, 103-124. BC940100S
