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

Abstract

The secondary structure of a recently identified ATP-binding RNA aptamer consists of apurine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked oneither side by helical stem segments. The ATP ligand targets the internal loop and bulgedomains, inducing a structural transition in this RNA segment on complex formation.Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugarhydroxyl 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 ofcompact RNA folds such as found in the AMP–RNA aptamer complex. One approachincorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeledadenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, withlabeling incorporation through chemical synthesis facilitated by generating the aptamer fromtwo separate strands. The unambiguous assignments deduced from the 15N labeling studieshave been verified from an independent labeling strategy where individual guanines in theinternal loop have been replaced, one at a time, by inosines and assignments were made onthe basis of the large 2 ppm downfield shift of the guanine imino protons on inosinesubstitution. The strengths and limitations of the inosine-for-guanine substitution approachemerge from our studies on the AMP–RNA aptamer complex. The assignment of theinternal loop and bulge imino and amino protons was critical in our efforts to define thesolution structure of the AMP–RNA aptamer complex since these slowly exchangingprotons exhibit a large number of long-range intramolecular NOEs within the RNA, as wellas intermolecular NOEs to the AMP in the complex. The current application of specific 15Nand inosine labeling approaches for exchangeable imino and amino proton assignments in thenonhelical segments of an RNA aptamer complex in our laboratory complements selective 2Hand 13C approaches to assign nonexchangeable base and sugar protons in RNA andligand–RNA complexes reported in the literature.