Accurate inference of the full base-pairing structure of RNA by deep mutational scanning and covariation-induced deviation of activity

Despite the transcription of noncoding RNAs in 75% of the human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained only from costly and inefficient 3D structure determination. Here, we performed deep mutational scanning of self-cleaving CPEB3 ribozyme by error-prone PCR and showed that a library of <5x104 single-to-triple mutants is sufficient to infer all 26 including nonhelical and noncanonical base pairs at the precision of a single false positive. The accurate inference, further confirmed by a twister ribozyme, is resulted from covariation analysis by utilizing both functional and nonfunctional variants for unsupervised learning, followed by restrained optimization. The result highlights the usefulness of deep mutational scanning for high-accuracy structural inference.