Molecular Immunology. Vol. 32, No. 14/I5, pp. 1057-1064. 1995 Pergamon Copyright (I! 1995Elsevier Science Ltd Printed in Great Britain. All rights reserved 0161-5890/95 $9.50+ 0.00 SPECIFICITY OF MONOCLONAL ANTIBODIES PRODUCED AGAINST PHOSPHOROTHIOATE AND RIB0 MODIFIED DNAs LAURA J. P. LATIMER, YEHENEW M. AGAZIE, RALPH P. BRAUN. KEN J. HAMPEL and JEREMY S. LEE* Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OWO Canada (First received 26 January 1995; accepted in revisedform 23 May 1995) Abstract-A large number of phosphorothioate DNAs and mixed ribo/deoxyribo duplexes were prepared and their immunogenicity was studied in mice. Only those polymers which were nuclease- resistant were immunogenic and in these cases monoclonal antibodies were prepared. The specificity of the antibodies was measured by direct and competitive Solid Phase Radioimmune Assay (SPRIA) and on this basis four types of antibody could be identified. Type I antibodies are specific for the immunizing polymer and show very limited crossreactivity. For example, Jel 384 binds only to poly(dsA).poly(dT); Jel 453 and 462 bind only to poly(dsG).poly(dC)and poly(dsG).poly(dm’C). Type II antibodies bind to most polymers containing the appropriate modification but will not bind to unmodified DNAs. For example, Je1343 binds to most thio DNAs regardless of sequence; Jel346 binds well to most ribose-containing polymers and may be a useful reagent for the detection of the ‘A’ family of conformations. Type III antibodies bind to most nucleic acids whether modified or not. Their specificities are similar to autoimmune antibodies. Type IV antibodies are single strand-specific such as Jel 383 which binds to poly(dT). There were no examples of antibodies which bound specifically to the immunizing DNA and the unmodified polymer. Thus, modified DNAs cannot be used to prepare sequence-specific reagents. Also, the immunogenicity of modified nucleic acids may limit their usefulness in antisense technologies. Key words: DNA-binding antibodies, immunogenicity, nuclease resistance. INTRODUCTION Nucleic acids with backbone modifications are widely used as antisense oligonucleotides (Wagner, 1994). This technology is a general method for influencing gene expression or viral replication by adding exogenous oli- gonucleotides which bind to a target sequence (Maher, 1992). For therapeutic applications the antisense oli- gonucleotide must be capable of penetrating the plasma membrane as well as being resistant to nucleases. Since most naturally-occurring DNA and RNA is rapidly degraded by intra-and extracellular nucleases, it is not suitable for antisense therapy (Eckstein, 1985). For this reason oligonucleotides have been prepared with back- bone modifications which render them nuclease-resistant. These modifications include u-nucleotides, methyl phos- phonates, peptide linkages and phosphorothioate (POS) groups (Gish and Eckstein, 1989; Nielsen et al., 1991; Cros et al., 1994; Xodo et al., 1994). However, unlike DNA and RNA, modified nucleic acids are immu- nogenic, probably for the very reason that they are nucle- ase-resistant and thus survive long enough to be recognized by the immune system (Braun and Lee, 1988). *Author to whom correspondence should be addressed. Therefore, lack of immunogenicity may have to be sac- rificed to achieve nuclease resistance. A second reason for studying the immunology of modi- fied nucleic acids is that the resultant antibodies may cross react with the unmodified form. In this way, sequence and structure-specific reagents could be prepared against antigens which are not normally immunogenic. For example, antibodies to Z DNA, triplexes or DNA/RNA hybrids have proved to be very useful for the detection and analysis of these unusual structures but, in general, sequence-specific antibodies are not available (Anderson et al., 1988; Stollar, 1991; Stollar, 1994). Previously, we prepared a series of POS DNAs and mixed ribo/ deoxyribo duplexes (Latimer et al., 1989; Braun and Lee. 1988). Some of these modified nucleic acids, particularly those with high GC content, were nuclease-resistant and immunogenic. Also, serum antibodies from mice immu- nized with poly[d(GsC)], for example, showed some bind- ing to the unmodified poly[d(GC)]. Thus, by immunizing with poly[d(GsC)] it might be possible to prepare mono- clonal antibodies which bound only to poly[d(GsC)] and poly[d(GC)]. For these reasons, mice have been immunized with a large number of POS DNAs and mixed ribo/deoxyribo duplexes. In those cases where the polymers were immu- 1057