Antibody•Nucleic Acid Complexes. Immunospecific Retention of Globin Messenger Ribonucleic Acid with Antibodies Specific for 7-Methylguanosine

Antibodies specific for 7-methylguanosine (m^G) were immobilized to Sepharose, and this adsorbent (antim⁷G/ Sepharose) was tested for its ability to select for globin mRNA on the basis of its 5'-terminal, m⁷G-containing cap, i.e., m⁷G(5')ppp(5')N. Preliminary studies with [³H]m⁷G indicated that the binding of ³H-labeled hapten to immobilized antibody was (i) essentially complete within 30 min, (ii) not affected significantly by variations in pH (5.0-9.0), temperature (0-56 °C), or salt concentrations (0.01-1.0 M NaCl), and (iii) specific for m7G-containing (oligo)nucleotides with no detectable cross-reactivity toward comparable unmethylated (oligo)nucleotides. Estimates based upon the amount of active antibody coupled to Sepharose (0.15 µg/µL) and its affinity constant (K_a = 5 X 10⁷ M^-1) revealed that 50 fiL of antim⁷G/ Sepharose was sufficient to quantitatively bind the equivalent of 5 fig of globin mRNA, a value in excellent agreement with that experimentally obtained. In assessment of the immunospecific selection of mRNA, rabbit globin mRNA was specifically labeled at each terminus via periodate oxidation and NaB³H₄ reduction. Upon recovery of oligo-(dT)-cellulose, the ³H-labeled mononucleotides derived from enzymatic digestion (P₁ nuclease and tobacco acid pyrophosphatase) of ³H-labeled globin mRNA yielded a [³H]- pm⁷G:[³H]pA ratio of 0.41. This result indicated the presence of 3'-terminal fragments of mRNA generated during the labeling reaction and enriched during recovery of oligo(dT)- cellulose chromatography. However, when incubated with anti-m⁷G/Sepharose, 63% of the poly(A)-containing -labeled globin mRNA was immunospecifically retained and possessed a [³H]pm⁷G:[³H]pA ratio of 0.95 in contrast to a 0.15 ratio for the nonretained preparation. Last, potential degradation of immunospecifically retained, unlabeled globin mRNA was not evident as evaluated by analysis of the resulting in vitro translational product, i.e., rabbit globin (ca. 15 000 molecular weight). Moreover, kinetic data from the latter experiments demonstrated that the translation of m7Gselected mRNA was 30-50% more efficient than that of unfractionated mRNA preparations. As determined from sucrose gradient centrifugation data, increased translational efficiency was attributed to the removal of an 18S rRNA contaminant.