Aromatic and basic residues play a key role at the NCL RBD3-4-miRNA interface
RNA-NCL docking scenarios of both RBD3-4 and RBD1-4 with miRNA models highlight the role of some of the positively charged arginine residues in β-2 (R291, R293) as well as the linker between β-2 and β-3 on RBD4 (R298) as these residues are consistently predicted to be involved in nearly all of the docking scenarios analyzed. These basic residues form salt bridges with the phosphate backbone of the various miRNAs. Similarly, our results indicate that aromatic residues on β-1 (F269 in RBD4) and β-3 (Y219 and F221 in RBD3 & F306 and F308 in RBD4) interact with miRNA molecules through π- π stacking and π-anion interactions with the nucleobases and the phosphate backbone, respectively. Importantly, the docking analysis of RBD1-4 models align with those of RBD3-4 models (Supporting Figure S3 ). Our results also show that residues on RNP motifs from RBD1-2 are largely not involved in NCL RBD-miRNA interactions, despite the similar functional profile of the RNP motifs. A control docking analysis using only RBD1-2 yields noisy scenarios with no clear trend (Supporting Figure S4 ), again reinforcing the idea that NCL RBD3-4 preferentially interact with the miRNA under study.
Based on the docking results, we show that NCL-RBDs interact with pri-miRNA nucleotides almost exclusively within the region that corressponds to the mature miRNA molecule. In our results, NCL is predicted to interact frequently with G-U pairs and kink turns (Supporting Figure S5 ). Interactions were observed predominantly at the major groove regions of the double stranded miRNA molecules as these regions were more accessible when compared to the minor groove regions.