Specific labeling approaches to guanine and adenine imino and amino proton assignments in the AMP-RNA aptamer complex.

The secondary structure of a recently identified ATP-binding RNA aptamer consists of a purine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked on either side by helical stem segments. The ATP ligand targets the internal loop and bulge domains, inducing a structural transition in this RNA segment on complex formation. Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugar hydroxyl chemical shift range are observed on AMP-RNA aptamer complex formation. This paper outlines site-specific labeling approaches to identify slowly exchanging imino (guanine) and amino (guanine and adenine) protons in internal loop and bulge segments of compact RNA folds such as found in the AMP-RNA aptamer complex. One approach incorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeled adenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, with labeling incorporation through chemical synthesis facilitated by generating the aptamer from two separate strands. The unambiguous assignments deduced from the 15N labeling studies have been verified from an independent labeling strategy where individual guanines in the internal loop have been replaced, one at a time, by inosines and assignments were made on the basis of the large 2 ppm downfield shift of the guanine imino protons on inosine substitution. The strengths and limitations of the inosine-for-guanine substitution approach emerge from our studies on the AMP-RNA aptamer complex. The assignment of the internal loop and bulge imino and amino protons was critical in our efforts to define the solution structure of the AMP-RNA aptamer complex since these slowly exchanging protons exhibit a large number of long-range intramolecular NOEs within the RNA, as well as intermolecular NOEs to the AMP in the complex. The current application of specific 15N and inosine labeling approaches for exchangeable imino and imino proton assignments in the nonhelical segments of an RNA aptamer complex in our laboratory complements selective 2H and 13C approaches to assign nonexchangeable base and sugar protons in RNA and ligand-RNA complexes reported in the literature.