The immunogenic properties of poly(dI).poly(dC) and poly(rI). poly(dC)--analysis by monoclonal antibodies.

Eight monoclonal antibodies were prepared from a C57/black mouse which had been immunized with poly(rI).poly(dC). Two of the antibodies were specific for the RNA-DNA duplex but bound about 100-fold better to poly(rI).poly(dC) than to poly(rA).poly(dT). The other six antibodies were single-strand specific and bound to poly(rI) and in most cases to poly(dI) as well, but not to other single-stranded nucleic acids. Similarly, 10 monoclonal antibodies were produced from mice immunized with poly(dI).poly(dC). Nine of these were specific for poly(dI) while the other had a strong preference for poly(dC). Thus, in contrast to poly(rI).poly(dC), the all DNA duplex poly(dI). poly(dC) only elicits duplex specific antibodies at very low frequency, if at all.

Functional groups on 'Z' DNA recognized by monoclonal antibodies.

Both brominated poly[d(GC)] and poly[d( Gm5C )] form stable left-handed Z-DNA structures at physiological ionic strengths. These two antigens were used to prepare monoclonal antibodies from immunized mice. The specificity of the antibodies was studied in detail with a solid-phase radioimmune assay as well as by means of competition experiments. Both immunogens produced several relatively non-specific antibodies but two types of very specific antibody were also distinguished. The first binds poly[d( Gm5C )] but not brominated poly[d(GC)] while the other has the opposite specificity and will only bind the brominated polymer.

A monoclonal antibody specific for the duplex DNA poly[d(TC)].poly[d(GA)].

Although most duplex DNAs are not immunogenic some synthetic DNAs such as poly[d(Tm5C)].poly[d(GA)] are weakly immunogenic allowing the production of monoclonal antibodies. The specificity of one of these antibodies, Jel 172, was investigated in detail by a competitive solid-phase radioimmune assay. Jel 172 bound well to poly[d(TC)].poly[d(GA)] but not to other duplex DNAs such as poly[d(TTC)].poly[d(GAA)] and poly[d(TCC)].poly[d(GGA)]. The binding to poly[d(Br5UC)].poly[d(GA)] was enhanced while that to poly[d(TC)].poly[d(IA)] was decreased compared to poly[d(TC)].poly[D(GA)]. Thus, not only is the antibody very specific for a sequence of duplex DNA but it also appears to recognize functional groups in both grooves of the helix.

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C).

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.

Poly(pyrimidine) . poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH.

Poly(pyrimidine) . poly(purine) tracts have been discovered in the 5'-flanking regions of many eucaryotic genes. They may be involved in the regulation of expression since they can be mapped to the nuclease-sensitive sites of active chromatin. We have found that poly(pyrimidine) . poly(purine) DNAs which contain 5-methylcytosine (e.g. poly[d(Tm5C)] . poly[d(GA)]) will form a triplex at a pH below 8. In contrast, the unmethylated analogue, poly[d(TC)] . poly[d(GA)] only forms a triplex at pHs below 6. Synthetic DNAs containing repeating trinucleotides and poly[d(Um5C)] . poly[d(GA)] behave in a similar manner. Thus the stability of a triplex can be controlled by methylation of cytosine. This suggests a model for the regulation of expression based upon specific triplex formation on the 5'-side of eucaryotic genes.

Characterization of monoclonal antibodies specific for isopentenyl adenosine derivatives occurring in transfer RNA.

A family of isopentenyl adenosine derivatives are naturally occurring components of transfer RNA and are involved in several different functional roles in the cell. To facilitate the study of the biochemistry of these modified nucleosides we have raised monoclonal antibodies to N6-(delta 2-isopentenyl)adenosine and N6-(4-hydroxy-3-methyl-but-2-enyl)adenosine. The antibodies show considerable specificity and three characteristic types are distinguishable. The first type have the hydroxylated derivative as the preferred antigen, the second type have isopentenyl adenosine as the preferred antigen and a third type show a specificity for all isopentenyl-containing derivatives.