ISOLATION AND CHARACTERIZATION OF A MONOCLONAL ANTI-QUADRUPLEX DNA ANTIBODY FROM AUTOIMMUNE VIABLE MOTH-EATEN MICE

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library deriv ed from 3-month-old nonimmunized autoimmune, immunodeficient ''viable motheaten'' mice. This antibody has been tested extensively in vitro a nd found to bind specifically to DNA quadruplex structures formed by t wo biologically relevant sequence motifs. Scatchard and nonlinear regr ession analyses using both one- and two-site models were used to deriv e association constants for the antibody-DNA binding reactions. In bot h cases, quadruplexes had higher association constants than tripler an d duplex molecules. The anti-quadruplex antibody binds to the quadrupl ex formed by the promoter-region-derived oligonucleotide d(CGCG(4)GCG) (K-a = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-deriv ed quadruplexes formed by the oligonucleotides d(TG(4)) and d(T(2)G(4) T(2)G(4)T(2)G(4)T(2)G(4)) (K-a = 5.38 x 10(6) and 1.66 x 10(7) M-1, re spectively). The antibody binds both types of quadruplexes but has pre ferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti- DNA quadruplex antibodies from anti-quadruplex antibody-producing tiss ue culture supernatants have at least 10-fold higher affinity for quad ruplexes than for tripler and duplex DNA structures of similar base co mposition and length. The antibody binds intramolecular DNA triplexes formed by d(G(4)T(3)G(4)T(3)C(4)) and d(C(4)T(3)G(4)T(3)G(4)), and the duplex d(CGCGCGCGCG)(2) with an affinities of 6.76 x 10(5), 5.59 x 10 (5), and 8.26 x 10(5) M-1, respectively. Competition experiments showe d that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadru plex is bound structure-specifically. To our knowledge, this is the fi rst immunological reagent known to specifically recognize quadruplex s tructures. Subsequent sequence analysis demonstrates homologies betwee n the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to contro l cell aging via telomeric DNA interactions. The presence of this anti body in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.