The Radioiodination of Ribopolymers for Use in Hybridizational and Molecular Analyses

Procedures have been developed for the preparation of radioiodinated RNA suitable for molecular characterization and hybridization. Incorporation of radioiodine into RNA in a thallic trichloride-catalyzed reaction is strongly pH-dependent. Above neutral pH little uptake of isotope into polynucleotide occurs, although incorporation into protein proceeds rapidly. The initial linkage of iodine incorporated into RNA at pH 5 is unstable and as much as 70% of the iodine originally associated with the polynucleotide can be dissociated by further heating above neutral pH. This behavior can be explained in terms of the reactions of the individual nucleotides. The predominant product formed with uridylate groups during reaction for 10 min at pH 5 is unstable and 95% of the adduct is rapidly released on heating at pH above 6. At lower pH the release is gradual but approaches the same final level of residual stable linkage. Cytidylate also forms an unstable iodine-containing intermediate. However, the initial addition product to polycytidylate was rapidly converted to a stable linkage throughout the pH range tested and underwent the transition even on standing at room temperature at pH 5. Purine groups in polynucleotides react to a lesser extent and composed less than 3% of the total isotope stabilized in RNA. When reactions were carried out under conditions which yield nucleic acid labeled to high specific activity, the iodination of residues in polyguanylate and also nucleotides present in polyadenylic acid was observed. Iodine retained in the respective purine residues after a reisolation procedure which lasted 48 hours was stable to heating at pH 4 to 9. The procedure for radioiodination and purification of the reaction product has been developed to allow experimentation with nucleic acids which are available in only small quantities. The physicochemical characterization of such RNAs or RNA fractions is complicated by the incorporation of iodine into protein, DNA, and other, undefined, contaminants. These substances can be eliminated from high molecular weight RNA by including a filtration step and sedimentation through cesium sulfate in the postlabeling workup of the iodinated RNA. The new procedures decreased the background level in hybridization reactions to 0.01 %. In addition to the base composition of RNA, our results suggest that secondary molecular structure of RNA may be important to the extent of reaction with iodine. This latter factor must be taken into consideration for interpretation of experiments comparing profiles of iodine-labeled nucleic acids. In the same regard, the results show that reaction conditions yielding highly labeled RNA do not cause chain breakages.