Inosine and N1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNAAla are major epitopes for anti-PL-12 myositis autoantibodies

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen), Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N-1-methylinosine (m(1)I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala), The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm(1)IpUp, wh ich corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m(1)I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm(1)Ip, are not antigen ic, Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N-1-methyl groups of N-1-methylinosine-37) within an anticodon loop structure of a tRNA molecule, We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N-1-methylinosine and N-1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oliganucleotides containing N-1-methylinosine and N-1-methyl-2'-deoxyinosine.