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 N1-methylinosine (m1I) 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 IpGpCpm1IpUp, which 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 m1I within the synthetic
polynucleotide. Synthetic
oligodeoxyribonucleotide analogs of the
tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. 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 N1-methyl groups of N1-methylinosine-37) within an
anticodon loop structure of a
tRNA molecule. We also describe the chemical synthesis of the
phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of
oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.