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RNA. 2003 Oct;9(10):1282-9.

Quantitation of free energy profiles in RNA-ligand interactions by nucleotide analog interference mapping.

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1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.

Abstract

RNA interactions with protein and small molecule ligands serve a wide variety of biochemical functions in the cell. To best understand the specificity and affinity of these interactions, the free energy contribution made by individual function groups in the RNA must be determined. As an efficient method for obtaining such energetic profiles, we report quantitative nucleotide analog interference mapping (QNAIM). This extension of the NAIM methodology uses the magnitude of analog interference as a function of ligand concentration to calculate binding constants for RNA with individual analog substitutions. In this way, QNAIM not only defines which functional groups are important to an interaction but simultaneously determines the energetic contribution made by each occurrence of that functional group within the RNA polymer. To establish the utility of this approach, QNAIM was used to quantify functional group interactions within the signal recognition particle (SRP), specifically the 4.5S RNA with the M domain of Ffh. In each of the cases in which energetic data were available from previous site-specific substitution analyses, QNAIM provided nearly equivalent results. These experiments on a model system demonstrate that QNAIM is an efficient method to establish a chemically detailed free energy profile for a wide variety of RNA-ligand interactions.

PMID:
13130142
PMCID:
PMC1370492
[Indexed for MEDLINE]
Free PMC Article
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