Bioconjugate Chem. 1990, 7, 187-191
tion by Complementary Oligonucleotides Covalently Linked to Intercalating Agents. Proc. Natl. Acad. Sci. U.S.A. 83, 1227-1231. (335) Cazenave, C., Loreau, N., Thuong, N. T., Toulme, J.-J., and Helene, C. (1987) Enzymatic Amplification of Translation Inhibition of Rabbit @-GlobinmRNA Mediated by Antimessenger OligodeoxynucleotidesCovalently Linked to Intercalating Agents. Nucleic Acids Res. 15, 4717-4735.
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(336) Verspieren, P., Cornelissen, A. W. C. A., Thuong, N. T., Helene, C., and Toulme, J.-J. (1987) An acridine-linked oligodeoxynucleotide targeted to the common 5’ end of trypanosome mRNAs kills cultured parasites. Gene 61, 307-315. (337) Zerial, A., Thuong, N. T., and Helene, C. (1987) Selective Inhibition of the Cytopathic Effect of Type A Influenza Viruses by Oligodeoxynucleotides Covalently Linked to an Intercalating Agent. Nucleic Acids Res. 15, 9909-9919.
TEACHING EDITORIAL A Brief Guide to Nucleic Acid Chemistry Paul S. Miller Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received January 19, 1990 STRUCTURE OF NUCLEIC ACIDS Nucleic acids encode the genetic information of all living organisms and are intimately involved in the conversion of this information into cellular proteins and enzymes. Cellular nucleic acids are polymeric molecules which are composed of three basic units: a base, a sugar, and a phosphodiester group. The arrangement of these three groups to form either DNA or RNA is shown in Figure 1. T h e nitrogenous, heterocyclic bases are derivatives of purine or pyrimidine. The four bases commonly found in DNA are adenine, guanine, cytosine, and thymine. These same bases are also found in RNA with the exception that thymine is replaced by uracil. In addition, a variety of modified bases, such as N 4,N4-dimethyladenine and N ‘-methylguanine are found in messenger RNA, transfer RNA, and ribosomal RNA. The bases are linked to 2’-deoxyribose in DNA or ribose in RNA via an N-glycosyl bond to form a nucleoside. The nucleosides are named according to the heterocyclic base which they contain. The nucleosides found in DNA are 2’-deoxyadenosine, 2’-deoxyguanosine, 2’-deoxycytidine, and thymidine. The nucleosides commonly found in RNA are adenosine, guanosine, cytidine, and uridine. 2’-0Methylribosyl nucleosides are also found in RNA, particularly in messenger RNA and ribosomal RNA. The nucleosides are linked together via phosphate ester groups to form the sugar phosphate backbone of the nucleic acid. Thus esterification of the 3’-hydroxyl of one nucleoside and the 5’-hydroxyl of the next nucleoside unit results in the formation of a 3‘-5‘ internucleotide bond. This type of linkage is found in both DNA and RNA. In the case of RNA the internucleotide bond could also extend from the 2’-hydroxyl to form a 2‘-5‘ internucleotide bond. Such 2’--5’ linkages are found in certain oligoadenylates which are synthesized in mammalian cells in response to interferon ( I ) . A single phosphorlyated nucleoside unit is called a nucleotide. T h e sequence of nucleotides within the nucleic acid chain determines the genetic information encoded by the nucleic acid. The purine bases, adenine and gua-
nine, can form hydrogen bonds with the pyrimidine bases, thymine (uracil in RNA) and cytosine, respectively (see Figure 2). The base pairs formed between these so-called complementary bases enable separate chains of nucleic acids to interact with one another. In DNA, separate nucleic acid strands form a double-helical structure in which the sugar phosphate backbones run in an antiparallel direction. Double-helical DNA, which usually exists in a right-handed, B-type conformation, can exist in a variety of conformational forms including left-handed helices (2). The particular conformation depends upon the nucleotide sequence and the environment of the DNA. DNA can also exist in a triple-stranded form in which three bases form a triad via hydrogen-bonding interactions as shown in Figure 2 (2, 3). Although RNA is often t h o u g h t of a s a singles t r a n d e d molecule, self-complementary nucleotide sequences present within the single strand give rise to the formation of intramolecular helical regions. These intramolecular interactions can produce a tremendous variety of helical and looped structural regions and account for the secondary structure within RNA molecules. In addition to these secondary structural features, further folding and hydrogen-bonding interactions between bases in remote parts of the molecule give rise to a tertiary structure. The combination of these interactions results in overall three dimensional structure, whose complexity approaches that found in proteins. This complexity has been most clearly revealed in the structure of transfer RNA ( 4 ) . Nucleic acid structure has been elucidated at the atomic level of resolution by nuclear magnetic resonance spectroscopy and X-ray diffraction techniques. In addition to studying nucleic acid structure, recent X-ray experiments have been used to examine the interactions of proteins with nucleic acids. For example, the structures of the complex formed between the restriction enzyme EcoR I and a deoxyribonucleotide duplex, and of the complex formed by glutaminyl tRNA with its cognate aminoacyl synthetase have been determined (5, 6). Such studies promise to lead to further insights into how nucleic acids
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Figure 2. Watson-Crick base-pairing interactions between adenine and thymine and guanine and cytosine (a) and triple-strand hydrogen-bonding interactions (b).
function as repositories of genetic information. CHEMISTRY OF NUCLEIC ACIDS Nucleic acids can undergo a wide variety of reactions with chemical reagents, both man-made and naturally occurring (7). These reactions can take place at the bases, the sugars, and the phosphodiester internucleotide linkage. T h e heterocyclic bases can be modified by both nucleophilic and electrophilic reagents (8). The exocyclic amino groups of adenine, guanine, and cytosine are subject to deamination reactions. Reaction at the 5,6double bonds of pyrimidines by nucleophiles such as hydrazine can ultimately lead to breakdown and loss of the pyrimidine ring. Alkylating agents such as dimethyl sulfate, alkylnitrosoureas, epoxides, and nitrogen and sulfur mustards can react with ring nitrogens as well as the exocyclic nitrogens and oxygens of the bases. In addition, a variety of natural products such as aflatoxin and
mitomycin react with the nucleic acid bases. Adduct formation can seriously modify or even prevent hydrogenbonding interactions between the bases which can ultimately lead to mutations and possibly to malignant cell transformation. Modification of the bases can also lead t o their excision from the nucleic acid. T h e N-glycosyl bond of deoxynucleosides in DNA is more labile than the corresponding bond in RNA. Treatment of DNA with acid, for example, results in loss of purine bases. Alkylation of adenine and guanine residues in DNA a t the N-3 or N-7 positions produces adducts which can undergo spontaneous depurination. The susceptibility of the bases to chemical modification depends to some extent upon the structure of the nucleic acid. Not surprisingly, single-stranded nucleic acids generally react more rapidly than double-stranded nucleic acids. In some cases the reactive site may be
Teaching Editorial
involved in hydrogen-bonding interactions with a complementary base and therefore is not subject to modification. The differential reactivity of single- vs doublestranded nucleic acids has been exploited to study the secondary and tertiary structure of both deoxyribo- and ribonucleic acids (9, 10). The nucleic acid bases can also undergo photochemical reactions (11). For example the 5,g-double bonds of adjacent thymine bases in the same strand of DNA can undergo a photocycloaddition reaction to form a cyclobutane adduct when DNA is irradiated with 254-nm UV light. Such adduct formation leads to distortions of the DNA helix. The sugars of RNA can undergo reaction with alkylating agents to form 2’-O-alkyl ethers. The sugars are also subject to radical induced abstraction reactions and subsequent degradation. Hydroxyl radicals produced as a result of ionization radiation or drugs which react via radical intermediates, such as the bleomycins and neocarzinostatin, can abstract hydrogen atoms from the C-l’, C-4’, or C-5’ positions of deoxyribose or ribose (12, 13). The sugar radicals which are produced as a result of these hydrogen-abstraction reactions undergo further oxidation and/or rearrangement which eventually leads to the destruction of the sugar skeleton and cleavage of the sugar phosphate backbone of the nucleic acid, producing strand breaks. The phosphodiester internucleotide linkage of RNA is sensitive to base-catalyzed hydrolysis in aqueous solution, whereas that of DNA is stable under alkaline conditions. The base lability of the RNA internucleotide bond is due to the presence of the 2’-hydroxyl group which can participate in an intramolecular attack on the phosphate group, resulting in hydrolysis of the phosphate internucleotide bond. The phosphodiester internucleotide bond can react with alkylating agents, resulting in the formation of alkylphosphotriesters (8). Phosphotriester formation creates a new chiral center in the nucleic acid backbone. Both R, and S, isomers are observed in these reactions. Alkyl phosphotriesters in DNA are stable to hydrolysis a t physiological pH, although they can be cleaved by treatment with strong acid or base. In contrast, alkylphosphotriester groups in RNA are subject to rapid hydrolysis, even a t neutral pH. This lability is due to intramolecular attack by the 2’-hydroxyl group of the sugar on the nonionic alkylphosphotriester group. Alkylphosphotriesters of oligonucleotides in which the 2’-OH is methylated are not subject to such hydrolysis (14). As described above, abasic sites can be created in nucleic acids by degradation of pyrimidine bases with hydrazine or by depurination with acid or alkylating agents. When treated with base, the sugar phosphate linkages a t these sites undergo @-eliminationreactions resulting in cleavage of the backbone. It has been possible to develop conditions under which abasic sites can be created in a basespecific manner. This ability has led to the development of chemical procedures which can be used to determine the nucleotide sequence of DNA and RNA (15, 16).
In addition to being the subject of various chemical reactions, recent experiments have shown that certain RNA molecules are capable of catalyzing hydrolysis, transesterification, and polymerization reactions of other nucleic acids (17,18). These catalytic RNAs are called ribozymes and appear to possess many of the catalytic properties previously associated only with enzymes. It appears that the catalytic properties of ribozymes are dependent upon
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both the sequence and the secondary and tertiary structures of the RNA molecule. CHEMICAL SYNTHESIS OF NUCLEIC ACIDS The ability to synthesize oligonucleotides and oligonucleotide analogues of defined nucleotide sequence provides materials for a variety of physical and biochemical studies. Both single-stranded and double-stranded oligonucleotides serve as convenient and manageable models for studies on the structure and conformation of nucleic acids. Oligonucleotides are used as primers for nucleic acid polymerizing enzymes and are used to construct artificial genes for the preparation of proteins by recombinant DNA techniques. In addition, oligonucleotides are used as probes to detect and characterize cellular nucleic acid sequences and as such are finding increasing use as clinical, diagnostic tools (19). Recent studies suggest that oligonucleotides and oligonucleotide analogues may be used to control gene expression in living cells and show considerable potential for eventual use as antiviral and therapeutic agents (20). Such oligomers have been termed antisense oligonucleotides. The chemical syntheses of oligonucleotides involve coupling a suitably protected nucleotide with another suitably protected nucleotide or oligonucleotide to form a 3’-5’ internucleotide linkage between the two units (21, 22). Protecting groups are required on the exocyclic amino groups of the bases to prevent them from reacting under the coupling conditions. The most commonly used groups are the benzoyl group for the protection of the exocyclic amino groups of adenine and cytosine and the isobutyryl group for the protection of the amino group of guanine (see Figure 3a). Other groups such as the amidine and phenoxyacetyl protecting groups have also been used (23, 24). These protecting groups remain in place during the course of the synthesis and are removed from the completed oligomer a t the end of the synthesis by treatment with aqueous ammonium hydroxide. The hydroxyl groups of the sugars are protected in order to direct the incoming nucleotide unit to give the 3’-5‘ linkage. The 5’-hydroxyl is usually protected with a dimethoxytrityl group which can be selectively removed by acid treatment during the course of the synthesis. The synthesis of oligoribonucleotides presents special problems due to the presence of the 2’-hydroxyl group. This group is generally protected with an acetal protecting group such as the 4-methoxytetrahydropyran-4-yl group or with dimethyl-tert-butylsilyl group (25, 26). These protecting groups remain attached throughout the synthesis and are usually removed after the base-protecting groups have been removed. The acetal groups are removed by treatment with acid whereas the silyl protecting group can be removed by treatment with tetra-n-butylammonium fluoride in tetrahydrofuran solution. Originally oligonucleotide syntheses were carried out in solution under anhydrous conditions employing nucleoside 3’- or 5’-monophosphates or phosphate diesters as synthons. The coupling reaction was effected by using a condensing reagent such as mesitylenesulfonyl chloride. These reactions, which required several hours to complete, resulted in the formation of oligomers containing phosphodiester or phosphotriester linkages. Because the coupling reactions were rather inefficient and were accompanied by the formation of a number of side products, time-consuming column chromatography was required after each coupling step to purify the desired product. The efficiency of oligonucleotide syntheses was considerably improved with the advent of coupling proce-
190 (a)
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tion in the presence of amines gives oligomers with phosphoramidate linkages (33). The coupling procedures described above enable the preparation of oligonucleotides on insoluble polymer supports (21,22). In this methodology the nucleoside which occurs at the 3’-end of the oligomer chain is attached via a linker arm to a controlled pore glass support. The trityl group is removed from the nucleoside by treatment with dichloroacetic acid in methylene chloride and the first coupling reaction is carried out. After the coupling/ oxidation step, excess reagent is removed by simply washing the support. The 5’-hydroxyl of any unreacted nucleoside is “capped” by acylation with acetic anhydride and the next round of detritylation and coupling is carried out. In this way the oligomer chain is built up one nucleotide at a time from the 3’- to the 5’-end. The entire procedure has been automated and a number of oligonucleotide synthesizers are commercially available which are capable of synthesizing oligomers over 100 nucleotides in length.
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ACKNOWLEDGMENT
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dures using trivalent phosphorus chemistry (27). Two procedures are generally employed as shown in Figure 3. Protected nucleoside phosphoramidite synthons (Figure 3b) readily react with the 5‘-hydroxyl of a nucleoside in the presence of tetrazole, which serves as both an acid catalyst for the reaction and forms a highly reactive nucleoside tetrazolide phosphinate intermediate (28,29). This coupling reaction, which proceeds to virtual completion in a matter of minutes at room temperature in the presence of a 10-20-fold excess of the phosphoramidite, yields a phosphite internucleotide linkage. The phosphite linkage is then oxidized quantitatively with aqueous iodine to give the 0-(cyanoethy1)phosphotriesterinternucleotide bond. The 0-cyanoethyl group remains in place during the course of the synthesis and is removed by the ammonium hydroxide treatment used to remove the baseprotecting groups. Protected nucleoside H-phosphonate synthons (Figure 3c) react with nucleoside 5’-hydroxyl groups when activated with a condensing agent such as pivaloyl chloride (30,31). This coupling reaction proceeds with a yield of approximately 95-97% at room temperature. The resulting H-phosphonate internucleotide bond is readily and quantitatively converted to the phosphodiester internucleotide linkage by oxidation with aqueous iodine solution. The H-phosphonate linkage is a very versatile intermediate because it can be used to prepare variety of oligonucleotide analogues. Thus, for example, oxidation in the presence of elemental sulfur produces oligomers containing a phosphorothioate linkage (32),whereas oxida-
(1) Brown, G. E., Lebleu, B., Kawakita, M., Shaila, S., Sen, G. C., and Lengyel, P. (1976) Increased Endonuclease Activity
in an Extract from Mouse Ehrlich Ascites Tumor Cells Which Had Been Treated with a Partially Purified Interferon Preparation: Dependence on dsRNA. Biochem. Biophys. Res. Commun. 69, 114-122. (2) Saenger, W. (1984) Principles of Nucleic Acid Structure Springer-Verlag, New York. (3) Wells, R. D., Collier, D. A,, Hanvey, J. C., Shimizu, M., and Wholrab, F. (1988) The Chemistry and Biology of Unusual DNA Structures Adopted by 0ligopurine.Oligopyrimidine Sequences. FASEB J . 2,2939-2949. (4) Kim, S.-H., Quigley, G. J., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl,J., and Rich, A. (1973) ThreeDimensionalStructure of Yeast PhenylalanineTransfer RNA Folding of the Polynucleotide Chain. Science 179, 285-288. (5) Grable, H. W., and Rosenberg, J. M. (1986) Structure of the DNA-EcoR I Endonuclease Recognition Complex at 3A. Science 234, 1526-1541. (6) Rould, M., Perona, J., Soll, D., and Steitz, T. (1989) Structure of E. coli Glutaminyl-tRNA Synthetase Complexed with tRNAglm and ATP at 2.8A Resolution. Science 246, 11351142. ( 7 ) Mizuno, Y. (1986) The Organic Chemistry of Nucleic Acids Elsevier, New York. (8) Singer,B., and Grunberger (1983)Molecular Biology ofMutagens and Carcinogens Plenum Press, New York. (9) Furlong, J. C., Sullivan, K. M., Murchie, A. I. H., Gough, G. W., and Lilley, D. M. J. (1989)Localized Chemical Hyperreactivity in Supercoiled DNA: Evidence for Base Unpairing in Sequences that Induce Low-Salt Cruciform Extrusion. Biochemistry 28, 2009-2017. (10) Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J.-P., and Ehresmann,B. (1987)Probing the Structure of RNAs in Solution. Nucleic Acids Res. 15, 9109-9128. (11) Wang, S. Y. (1976) Photochemistry and Photobiology of Nucleic Acids Vol. I and 11, Academic Press, New York. (12) Kozarich,J., Worth, L., Jr., Frank, B., Christner, D., Vanderwall, D., and Stubbe,J. (1989)Sequence-Specific Isotope Effects on the Cleavage of DNA by Bleomycin. Science 245, 13961399. (13) Lee, S. H., and Goldberg, I. H. (1989) Sequence-Specific, Strand-Selective, and Directional Binding of Neocarzinostatin Chromophore to Oligodeoxyribonucleotides. Biochemistry 28, 10109-10116. (14) Miller, P. S., Braiterman, L. T., and Ts’o, P.O.P. (1977) Effects of a Trinucleotide Ethyl Phosphotriester, Gmp(Et)-
Bioconjugate Chem. 1990, 1,
Gmp(Et)U, on Mammalian Cells in Culture. Biochemistry 16, 1988-1996. (15) Maxam, A. M., and Gilbert, W. (1980) Sequencing EndLabeled DNA with Base-Specific Chemical Cleavages. Methods Enzymol. 65,499-560. (16) Peattie, D. (1979) Direct Chemical Method for Sequencing RNA. Proc. Natl. Acad. Sci. U.S.A. 76, 1760-1764. (17) Cech, T. (1987) The Chemistry of Self-splicing RNA and RNA Enzymes. Science 236, 1532-1539. (18) Uhlenbeck, 0. (1989) A Small Catalytic Oligoribonucleotide. Nature 328, 596-600. (19) Wallace, R. B., Studencke, A. B., and Murasugi, A. (1985) Biochimie 67, 755-762. (20) Cohen, J. S. (1989)Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression. Topics in Molecular and Structural Biology Vol. 12, MacMillan Press, London. (21) M. J. Gait, D. (1984) Oligonucleotide Synthesis, a Practical Approach IRL Press, Oxford. (22) Engels, J. W., and Uhlmann, E. (1989) Gene Synthesis. Angew. Chem., Int. Ed. Engl. 28, 716-734.
(23) McBride, L. J., Kierzek, R., Beaucage, S. L., and Caruthers, M. H. (1986) Amidine Protecting Groups for Oligonucleotide Synthesis. J . Am. Chem. SOC. 108, 2040-2048. (24) Wu, T., Ogilvie, K. K., and Pon, R. T. (1989) Prevention of Chain Cleavage in the Chemical Synthesis of 2’-Silylated Oligoribonucleotides. Nucleic Acids Res. 17, 3501-3517. (25) Lehmann, C., Xu, Y.-Z., Christodoulou, C., Tan, Z.-K., and Gait, M. J. (1989) Solid Phase Synthesis of Oligoribonucleotides Using 9-Fluorenylmethoxycarbonyl (Fmoc) for 5’-Hydroxyl Protection. Nucleic Acids Res. 17, 2379-2390. (26) Ogilvie, K. K., Usman, N., Nicoghosian, K., and Ceder-
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gren, R. J. (1988) Total Chemical Synthesis of a 77-Nucleotide-Long RNA Sequence Having Methionine-Acceptance Activity. Proc. Natl. Acad. Sci. U.S.A. 85, 5764-5768. (27) Letsinger, R. L., Finnan, J. L., Heavner, G. A., and Lunsford, W. B. (1975) Phosphite Coupling Procedure for Generating Internucleotide Links. J . Am. Chem. SOC.97, 32783279. (28) McBride, L. J., and Caruthers, M. H. (1983) An Investigation of Several Deoxynucleoside Phosphoramidites Useful for Synthesizing Deoxyoligonucleotides. Tetrahedron Lett. 24, 245-248. (29) Sinha, N., Biernat, J., McManus, J., and Koster, H. (1984) Polymer Support Oligonucleotide Synthesis XVIII: Use of (3-Cyanoethyl-N,N-dialkylamino-N-morpholinoPhosphoramidite of Deoxynucleosides for the Synthesis of DNA Fragments Simplifying Deprotection and Isolation of the Final Product. Nucleic Acids Res. 12, 4539-4557. (30) Garegg, P. J., Regberg, T., Stawinski, J., and Stromberg, R. (1987) Studies on the Synthesis of Oligonucleotides via the Hydrogenphosphonate Approach. Nucleosides Nucleotides 6, 283-286. (31) Froehler, B. C., and Matteucci, M. D. (1987) The Use of Nucleoside H-Phosphonates in the Synthesis of Deoxyoligonucleotides. Nucleosides Nucleotides 6, 287-291. (32) Stec, W. J., Zon, G., Egan, W., and Stec, B. (1984) Automated Solid Phase Synthesis, Separation, and Stereochemistry of Phosphorothioate Analogues of Oligodeoxyribonucleotides. J. Am. Chem. SOC.108, 6077-6079. (33) Froehler, B., Ng, P., and Matteucci, M. (1988) Phosphoramidate Analogues of DNA: Synthesis and Thermal Stability of Heteroduplexes. Nucleic Acids Res. 16, 4831-4839.
ARTICLES Labeling of Human IgG with Rhodium-105 Using a New Pentadentate Bifunctional Ligand M. R. A. Pillai,* C. S. John,? and D. E. Troutner Department of Chemistry, University of Missouri, Columbia, Missouri 65211. Received September 25, 1989
We report the labeling of human gamma globulin with the lo5Rh complex of a new pentadentate bifunctional ligand, 1,7-bis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine. Complexes of this ligand with lo6Rh were prepared by refluxing rhodium carrier spiked with lo5Rh at pH 9 in bicarbonate buffer. T h e complex was treated with a n excess concentration of thiophosgene t o prepare the isothiocyanate derivative which was extracted into CHCls. T h e CHC13 extract was dried and dissolved in DMF and reacted with a borate solution of human gamma globulin. Labeling yields were generally high and varied from 73% t o 93%, depending upon the concentration of human gamma globulin and the isothiocyanate derivative of the complex used. The overall recovery of rhodium activity varied from 59% t o 75% without taking into account activity lost due t o decay. The conjugation reaction was complete by 4 h. From 0.4t o 8.5 atoms of Rh could be incorporated per molecule of protein by this method. The activated isothiocyanate complex did not show any degradation when stored at room temperature for u p t o 4 days and then used for conjugation. Because of its ideal radionuclidic properties, lo5Rhcan be developed as a radiotherapeutic isotope (I). We have
* permanentaddress: Isotope ~ i ~ i ~ i B.A.R.c., ,,~, ~ 400 085, India. + Radiopharmaceutical Chemistry, George Washington University, 2300 I Street, N.W., Washington, DC 20037.
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demonstrated t h a t lo5Rh can be attached t o proteins through bifunctional chelating agents and that these methods are suitable for the labeling of antibodies with specific for radioimmunotherapy (2, ~ activities b ~high enough y 3). I n those studies we used tridentate bifunctional ligands derived from diethylenetriamine. One of our aims has been the development of a bifunctional ligand capable
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