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[175] Preparation of nucleoside-specific synthetic antigens
900
IMMUNOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[175]
[175] P r e p a r a t i o n of N u c l e o s i d e - S p e c i f i c S y n t h e t i c A n t i g e n s
By MICHAEL SELA and
HANNA UNGAR-WARON
One approach to the elucidation of immunological properties of nucleic acids consists of efforts to bind, chemically, their components to well-defined macromolecules, and to study the immunogenicity of such conjugates as well as the antigenic specificity of antibodies elicited by them. The catalytic oxidation of nucleosides yields nueleoside-5'-carboxylie acids/ which may be conjugated by means of N,N'-dieyclohexylearbodiimide to the terminal amino groups of synthetic multichain polypeptides. 2,~ This kind of conjugation keeps intact the positions 2' and 3' of the sugar moieties. The attachment of nucleoside-5'-carboxylic acids to the nonantigenie multichain poly-DL-alanyl-poly-L-lysine4 yields completely synthetic antigens such as uridine-pDLAla-pLLys~ (Fig. 1), which elicit upon injection into rabbits antibodies with specificity directed toward each of the attached nucleosides. ~ Thus the attachment of thymidine-5'-carboxylic acid to multichain p01y-DL-alanyl-poly-L-lysine results in an immunogen which produces highly specific antibodies in rabbits. These antibodies do not react with uridine, but their homologous reaction is strongly inhibited by deoxyuridine. This difference shows that the antibodies formed may distinguish between the ribose and deoxyribose moieties of the nucleosides, even though the sugars by themselves are devoid of any inhibitory effect in the homologous system. The antigenic specificity of the antibodies obtained may be defined either by the structure of the entire nucleoside attached, as in the case of uridine (where uracil does not inhibit the homologous reaction), or it may be due partly to its constituent base, as in the case of thymidine and adenosine, where thymine and adenine cross-react in their respective nucleoside systems. The nature of the linkage between base and sugar contributes too to the specificity of the antibodies obtained by this technique. Thus antibodies to uridine do not cross react with pseudouridine. Native calf thymus DNA does not react with antisera against the polymers containing uridine, thymidine, or adenosine but cross-precipitates with them after heat denaturation, or even better when denaturation is performed in the presence of formaldehyde. 1G. P. Moss, C. B. Reese, K. Schofield, R. Shapiro, and Lord A. R. Todd, J. Chem. Soc. p. 1149 (1963). M. Sela, H. Ungar-Waron, and Y. Shechter, Proc. Natl. Acad. Sci. U.S. 525 285
(1964). 3M. Sela and H. Ungar-Waron, Federation Proc. 24, 1438 (1965). ' M. Sela, S. Fuehs, and R. Amon, Bioche.m.J. 85, 223 (1962).
[175]
NUCLEOSIDE-SPECIFIC SYNTHETIC
6
~
q
901
ANTIGENS
" UridJne
"
OL-- Ala
L -
Lys
% Fro. 1. Schematic presentation of the multichain nucleoside-polypeptide conjugate uridine-pDLAla-pLLys (see text footnote 2).
Antithymidine serum does not cross-react with RNA as expected from its lack of reactivity with uridine. Heat-denatured E. coli RNA reacts both with antiuridine and antiadenosine sera after proper removal of serum RNase activity either by treatment with anti-RNase T-globulins or by repeated passages of the antibodies through Ambertite IRC-50 columns2 Preparation of Thymidine-S'-Carboxylic Acid 1 Thymidine (0.785 g, 3.25 millimoles) is dissolved in 120 ml of bicarbonate buffer, pH 9 (0.275 g, 3.25 millimoles, of sodium hydrogen carbonate and 0.12 g of sodium carbonate decahydrate). Platinum oxide is first reduced by hydrogenation in glacial acetic acid and then added (0.52 g) as catalyst to the buffered thymidine solution. Oxygen is bubbled into the rapidly stirred suspension maintained at 80 ° for 10 hours. Catalyst is removed by filtration (it can be reused) and the filtrate concentrated to 50 ml under reduced pressure, treated with Amberlite IR-120 (H ÷ form) and further evaporation to dryness. Additional concentration before Amberlite treatment brings about the precipitation of the reaction product on the resin, which should be avoided. Upon recrystallization a white crystalline product is obtained: melting point with decomposition 263-265°; R~, 0.50 in isopropanol-ammonia-water 7:1:2
902
IMMUNOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[175]
(v/v) and 0.66 in butanol-acetic acid-water 5:2:3 (v/v), descending chromatography; ~'max (in water) 267 m# (log c, 3.97). Preparation of Adenosine-5~-Carboxylic Acid 1 Adenosine (0.885 g, 3 millimoles) is dissolved in 110 ml of bicarbonate (3 millimoles) buffer, pH 9. Platinum catalyst (0.88 g) is added and oxygen is bubbled through the stirred suspension at 90 ° . After 40 hours the reaction is only half completed, but it is then discontinued in order to avoid accumulation of decomposition by-products. Catalyst is removed and the filtrate is concentrated to 10 ml and brought to neutral pH by the addition of dilute sulfuric acid. It is then charged on a DEAE-cellulose column (1.8 X 40) equilibrated at pH 7.0. The unreacted adenosine is eluted with water, while the adenosine-5'-carboxylic acid is retained by the resin. It is recovered from the column by elution with 0.05 M sodium hydroxide. The addition of dilute sulfuric acid to the fractions containing the sodium salt of adenosine-5'-carboxylic acid brings about the separation of the latter in the form of needles: decomposition point 320°; Rs, 0.35 in isopropanol-ammonia-water 7:1:2 (v/v) and 0.39 in butanol-acetic acid-water 5:2:3 (v/v), descending chromatography: hm,x (in water) 258 m~ (log ¢, 4.18). Preparation of the Nucleoside Conjugate of Multichain Poly-DL-Alanine 8 (Fig. 1) Multichain poly-nL-alanine (poly-DL-alanyl-poly-L-lysine, 1.5 g) is dissolved in 10 ml of water and mixed with a solution of 3 millimoles of nucleoside-5'-carboxylic acid in 180 ml of dimethyl formamide (the final reaction mixture should not contain more than 5% water). N,N'-Dicyclohexylcarbodiimide (DCC, 0.62 g, 3 millimoles) in 10 ml of dimethyl formamide is then added, and the reaction mixture is left overnight at room temperature. The reaction product is dialyzed against several changes of 0.05 M sodium hydrogen carbonate to eliminate the unreacted nucleoside-5'-carboxylic acids, which are sparsely water soluble, and then against distilled water. The precipitates formed in the dialysis bags (due to insoluble DCC and to urea derivatives formed) are removed by filtration and discarded, and the filtrate is dialyzed again, freeze-dried and stored at 2 ° . The nucleoside-CO content of the polymer is determined from the extinction at hm,x of the respective nucleoside, the multichain pOly-DLalanine by itself not contributing to the extinction in the region of UV absorption of the nucleosides. The amount of nucleoside-CO attached is in the range of 8-10% of the polymeric eoniugate obtained. Molecular weights, obtained from sedimentation and diffusion data, depend on the
[176]
DETECTION OF RNA BY AGAR DIFFUSION
903
molecular weight of the multichain poly-DL-alanine used in the synthesis of the conjugate, and are usually in the range of 80,000-120,000. M e t h o d of I m m u n i z a t i o n
The nucleoside conjugates of the synthetic polymer pDLAla-pLLys were injected into groups of four to eight rabbits. Immunization is carried out using an emulsion of equal volumes of 2.5% antigen in 0.9?'o sodium chloride solution and complete Freund's adjuvant. Upon each injection the animals receive 10 mg of antigen administered intramuscularly in their hind legs. Three to four injections are given at intervals of 10 days. The animals are bled weekly after immune response is obtained, and the antiserum is pooled and kept frozen until used. [ 176] I m m u n o l o g i c a l D e t e c t i o n of R i b o n u c l e i c A c i d s by Agar Diffusion
By F. LACOUR I m m u n e Sera
While purified RNA and their constituents are very poor immunogens, complexes which contain RNA, whether natural such as ribosomes, or artificial as obtained by adsorption to or conjugation with foreign proteins or polypeptides, give a much better immunological response. Thus, antibodies reacting with purified RNA could be demonstrated in some antiribosomal sera obtained upon immunization with bacterial ribosomes I or ribosomes from mouse ascitic cells3 With transfer RNA adsorbed to methylated serum albumin (MBSA) anti-RNA antibodies could be obtained? Uridine-specific antibodies produced following immunization with uridine conjugated to polypeptides4 or to bovine serum albumin ~ were also shown to cross-react with RNA. Preparation of M e t h y l a t e d B o v i n e Serum Albumin ~
Suspend 5 grams albumin in 500 ml absolute methyl alcohol and add 4.2 ml of 12N HC1. The protein dissolves and eventually precipitates again. Allow the mixture to stand in the dark for 3 days or more, occaE. Barbu and J. Panijel, Compt. Rend. Acad. Sci. 250, 1382 (1960). F. Lacour, J. Harel, L. Harel, and E. Nahon, Compt. Rend. Acad. Sci. 25S, 2322 (1962). 30. J. Plescia, N. C. Palczuk, W. Braun, and E. Cora-Figueroa, Science 148, 1102 (1965). 4 M. Sel~, H. Ungar-Waron and Y. Schechter, Proc. Natl. Acad. Sci. U.S. $2, 285 (1964). 5 M. H. Karol and S. W. Tanenbaum, Proc. Natl. Acad. Sci. U.S. 57, 713 (1967). ~J. D. Mandell and A. D. Hershey, Anal. Biochem. 1, 66 (1960).
IMMUNOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[175]
[175] P r e p a r a t i o n of N u c l e o s i d e - S p e c i f i c S y n t h e t i c A n t i g e n s
By MICHAEL SELA and
HANNA UNGAR-WARON
One approach to the elucidation of immunological properties of nucleic acids consists of efforts to bind, chemically, their components to well-defined macromolecules, and to study the immunogenicity of such conjugates as well as the antigenic specificity of antibodies elicited by them. The catalytic oxidation of nucleosides yields nueleoside-5'-carboxylie acids/ which may be conjugated by means of N,N'-dieyclohexylearbodiimide to the terminal amino groups of synthetic multichain polypeptides. 2,~ This kind of conjugation keeps intact the positions 2' and 3' of the sugar moieties. The attachment of nucleoside-5'-carboxylic acids to the nonantigenie multichain poly-DL-alanyl-poly-L-lysine4 yields completely synthetic antigens such as uridine-pDLAla-pLLys~ (Fig. 1), which elicit upon injection into rabbits antibodies with specificity directed toward each of the attached nucleosides. ~ Thus the attachment of thymidine-5'-carboxylic acid to multichain p01y-DL-alanyl-poly-L-lysine results in an immunogen which produces highly specific antibodies in rabbits. These antibodies do not react with uridine, but their homologous reaction is strongly inhibited by deoxyuridine. This difference shows that the antibodies formed may distinguish between the ribose and deoxyribose moieties of the nucleosides, even though the sugars by themselves are devoid of any inhibitory effect in the homologous system. The antigenic specificity of the antibodies obtained may be defined either by the structure of the entire nucleoside attached, as in the case of uridine (where uracil does not inhibit the homologous reaction), or it may be due partly to its constituent base, as in the case of thymidine and adenosine, where thymine and adenine cross-react in their respective nucleoside systems. The nature of the linkage between base and sugar contributes too to the specificity of the antibodies obtained by this technique. Thus antibodies to uridine do not cross react with pseudouridine. Native calf thymus DNA does not react with antisera against the polymers containing uridine, thymidine, or adenosine but cross-precipitates with them after heat denaturation, or even better when denaturation is performed in the presence of formaldehyde. 1G. P. Moss, C. B. Reese, K. Schofield, R. Shapiro, and Lord A. R. Todd, J. Chem. Soc. p. 1149 (1963). M. Sela, H. Ungar-Waron, and Y. Shechter, Proc. Natl. Acad. Sci. U.S. 525 285
(1964). 3M. Sela and H. Ungar-Waron, Federation Proc. 24, 1438 (1965). ' M. Sela, S. Fuehs, and R. Amon, Bioche.m.J. 85, 223 (1962).
[175]
NUCLEOSIDE-SPECIFIC SYNTHETIC
6
~
q
901
ANTIGENS
" UridJne
"
OL-- Ala
L -
Lys
% Fro. 1. Schematic presentation of the multichain nucleoside-polypeptide conjugate uridine-pDLAla-pLLys (see text footnote 2).
Antithymidine serum does not cross-react with RNA as expected from its lack of reactivity with uridine. Heat-denatured E. coli RNA reacts both with antiuridine and antiadenosine sera after proper removal of serum RNase activity either by treatment with anti-RNase T-globulins or by repeated passages of the antibodies through Ambertite IRC-50 columns2 Preparation of Thymidine-S'-Carboxylic Acid 1 Thymidine (0.785 g, 3.25 millimoles) is dissolved in 120 ml of bicarbonate buffer, pH 9 (0.275 g, 3.25 millimoles, of sodium hydrogen carbonate and 0.12 g of sodium carbonate decahydrate). Platinum oxide is first reduced by hydrogenation in glacial acetic acid and then added (0.52 g) as catalyst to the buffered thymidine solution. Oxygen is bubbled into the rapidly stirred suspension maintained at 80 ° for 10 hours. Catalyst is removed by filtration (it can be reused) and the filtrate concentrated to 50 ml under reduced pressure, treated with Amberlite IR-120 (H ÷ form) and further evaporation to dryness. Additional concentration before Amberlite treatment brings about the precipitation of the reaction product on the resin, which should be avoided. Upon recrystallization a white crystalline product is obtained: melting point with decomposition 263-265°; R~, 0.50 in isopropanol-ammonia-water 7:1:2
902
IMMUNOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[175]
(v/v) and 0.66 in butanol-acetic acid-water 5:2:3 (v/v), descending chromatography; ~'max (in water) 267 m# (log c, 3.97). Preparation of Adenosine-5~-Carboxylic Acid 1 Adenosine (0.885 g, 3 millimoles) is dissolved in 110 ml of bicarbonate (3 millimoles) buffer, pH 9. Platinum catalyst (0.88 g) is added and oxygen is bubbled through the stirred suspension at 90 ° . After 40 hours the reaction is only half completed, but it is then discontinued in order to avoid accumulation of decomposition by-products. Catalyst is removed and the filtrate is concentrated to 10 ml and brought to neutral pH by the addition of dilute sulfuric acid. It is then charged on a DEAE-cellulose column (1.8 X 40) equilibrated at pH 7.0. The unreacted adenosine is eluted with water, while the adenosine-5'-carboxylic acid is retained by the resin. It is recovered from the column by elution with 0.05 M sodium hydroxide. The addition of dilute sulfuric acid to the fractions containing the sodium salt of adenosine-5'-carboxylic acid brings about the separation of the latter in the form of needles: decomposition point 320°; Rs, 0.35 in isopropanol-ammonia-water 7:1:2 (v/v) and 0.39 in butanol-acetic acid-water 5:2:3 (v/v), descending chromatography: hm,x (in water) 258 m~ (log ¢, 4.18). Preparation of the Nucleoside Conjugate of Multichain Poly-DL-Alanine 8 (Fig. 1) Multichain poly-nL-alanine (poly-DL-alanyl-poly-L-lysine, 1.5 g) is dissolved in 10 ml of water and mixed with a solution of 3 millimoles of nucleoside-5'-carboxylic acid in 180 ml of dimethyl formamide (the final reaction mixture should not contain more than 5% water). N,N'-Dicyclohexylcarbodiimide (DCC, 0.62 g, 3 millimoles) in 10 ml of dimethyl formamide is then added, and the reaction mixture is left overnight at room temperature. The reaction product is dialyzed against several changes of 0.05 M sodium hydrogen carbonate to eliminate the unreacted nucleoside-5'-carboxylic acids, which are sparsely water soluble, and then against distilled water. The precipitates formed in the dialysis bags (due to insoluble DCC and to urea derivatives formed) are removed by filtration and discarded, and the filtrate is dialyzed again, freeze-dried and stored at 2 ° . The nucleoside-CO content of the polymer is determined from the extinction at hm,x of the respective nucleoside, the multichain pOly-DLalanine by itself not contributing to the extinction in the region of UV absorption of the nucleosides. The amount of nucleoside-CO attached is in the range of 8-10% of the polymeric eoniugate obtained. Molecular weights, obtained from sedimentation and diffusion data, depend on the
[176]
DETECTION OF RNA BY AGAR DIFFUSION
903
molecular weight of the multichain poly-DL-alanine used in the synthesis of the conjugate, and are usually in the range of 80,000-120,000. M e t h o d of I m m u n i z a t i o n
The nucleoside conjugates of the synthetic polymer pDLAla-pLLys were injected into groups of four to eight rabbits. Immunization is carried out using an emulsion of equal volumes of 2.5% antigen in 0.9?'o sodium chloride solution and complete Freund's adjuvant. Upon each injection the animals receive 10 mg of antigen administered intramuscularly in their hind legs. Three to four injections are given at intervals of 10 days. The animals are bled weekly after immune response is obtained, and the antiserum is pooled and kept frozen until used. [ 176] I m m u n o l o g i c a l D e t e c t i o n of R i b o n u c l e i c A c i d s by Agar Diffusion
By F. LACOUR I m m u n e Sera
While purified RNA and their constituents are very poor immunogens, complexes which contain RNA, whether natural such as ribosomes, or artificial as obtained by adsorption to or conjugation with foreign proteins or polypeptides, give a much better immunological response. Thus, antibodies reacting with purified RNA could be demonstrated in some antiribosomal sera obtained upon immunization with bacterial ribosomes I or ribosomes from mouse ascitic cells3 With transfer RNA adsorbed to methylated serum albumin (MBSA) anti-RNA antibodies could be obtained? Uridine-specific antibodies produced following immunization with uridine conjugated to polypeptides4 or to bovine serum albumin ~ were also shown to cross-react with RNA. Preparation of M e t h y l a t e d B o v i n e Serum Albumin ~
Suspend 5 grams albumin in 500 ml absolute methyl alcohol and add 4.2 ml of 12N HC1. The protein dissolves and eventually precipitates again. Allow the mixture to stand in the dark for 3 days or more, occaE. Barbu and J. Panijel, Compt. Rend. Acad. Sci. 250, 1382 (1960). F. Lacour, J. Harel, L. Harel, and E. Nahon, Compt. Rend. Acad. Sci. 25S, 2322 (1962). 30. J. Plescia, N. C. Palczuk, W. Braun, and E. Cora-Figueroa, Science 148, 1102 (1965). 4 M. Sel~, H. Ungar-Waron and Y. Schechter, Proc. Natl. Acad. Sci. U.S. $2, 285 (1964). 5 M. H. Karol and S. W. Tanenbaum, Proc. Natl. Acad. Sci. U.S. 57, 713 (1967). ~J. D. Mandell and A. D. Hershey, Anal. Biochem. 1, 66 (1960).