A procedure to validate and correct the ¹³C chemical shift calibration of RNA datasets

Abstract

Chemical shifts reflect the structural environment of a certain nucleus and can be used to extract structural and dynamic information. Proper calibration is indispensable to extract such information from chemical shifts. Whereas a variety of procedures exist to verify the chemical shift calibration for proteins, no such procedure is available for RNAs to date. We present here a procedure to analyze and correct the calibration of ¹³C NMR data of RNAs. Our procedure uses five ¹³C chemical shifts as a reference, each of them found in a narrow shift range in most datasets deposited in the Biological Magnetic Resonance Bank. In 49 datasets we could evaluate the ¹³C calibration and detect errors or inconsistencies in RNA ¹³C chemical shifts based on these chemical shift reference values. More than half of the datasets (27 out of those 49) were found to be improperly referenced or contained inconsistencies. This large inconsistency rate possibly explains that no clear structure–¹³C chemical shift relationship has emerged for RNA so far. We were able to recalibrate or correct 17 datasets resulting in 39 usable ¹³C datasets. 6 new datasets from our lab were used to verify our method increasing the database to 45 usable datasets. We can now search for structure–chemical shift relationships with this improved list of ¹³C chemical shift data. This is demonstrated by a clear relationship between ribose ¹³C shifts and the sugar pucker, which can be used to predict a C2′- or C3′-endo conformation of the ribose with high accuracy. The improved quality of the chemical shift data allows statistical analysis with the potential to facilitate assignment procedures, and the extraction of restraints for structure calculations of RNA.