Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: A site-directed spin labeling study

The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is b elieved to be one of the most frequently occurring tertiary interaction mot ifs in RNAs, but an isolated tetraloop/receptor complex has not been identi fied in solution. In the present work, site-directed spin labeling is appli ed to detect tetraloop/receptor complex formation and estimate the free ene rgy of interaction. For this purpose, the GAAA tetraloop/receptor interacti on was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA tetraloop. Formation of the tetraloop/receptor complex was monitored throu gh changes in the rotational correlation time of the tetraloop and the atta ched nitroxide. Results show that a hairpin containing the GAAA tetraloop f orms a complex with an RNA containing the Il-nucleotide GAAA tetraloop rece ptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 m M MgCl2, K-d = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the tetr aloop/receptor interaction in the absence of other tertiary constraints. Th e experimental strategy presented here should have broad utility in quantif ying weak interactions that would otherwise be undetectable, for both nucle ic acids and nucleic acid -protein complexes.