Structural Determination of the Dicer-2•R2D2 Complex Helen M. Donelick, Peter S. Shen, and Brenda L. Bass University of Utah. Salt Lake City, Utah, USA In Drosophila melanogaster, Dicer-2 is essential for multiple RNA interference functions, including small interfering RNA (siRNA) biogenesis. These siRNAs are 21-23 nucleotides in length (Elbashir et al., 2001), and one strand is loaded onto Argonaute-2 to form the active RNA-induced silencing complex (RISC). Loading the guide strand onto Argonaute-2 requires the formation of the RISC loading complex, which includes Dicer-2, siRNA, and R2D2, a dsRNA binding protein. R2D2 appears to interact with the Dicer-2 helicase domain (Nishida et al., 2013). The Dicer-2•R2D2 complex binds tightly to siRNA, and without R2D2, siRNAs are generated but cannot be passed on to Argonaute-2; therefore, R2D2 is essential for functional RNA interference (Liu et al., 2003, 2006). Although the RISC loading complex is vital for RNA interference, little is known about how this complex passes the guide strand, and no structural information exists on how the complex is formed. It has been suggested that the helicase domain of many Dicer proteins is the domain where accessory proteins can bind and modulate function (Hansen et al., 2020). A high-resolution structure of this complex will provide details into how R2D2 modulates the function of Dicer-2, which can then be validated with biochemical techniques. To acquire a structure, we built on cryo-EM work performed by my co-mentors. We performed single-particle analysis on a dataset collected on the University of Utah Titan Krios. The complex consisted of Dicer-2•R2D2, siRNA, and ATP. Our preliminary analysis of this structure shows an extra density on the helicase domain, not accounted for by an apo-Dicer-2 model. It appears this extra density on the helicase domain is R2D2, but the current resolution is still low; thus, exactly how this accessory factor regulates Dicer-2 remains unknown. We are continuing to analyze our dataset and utilize Topaz (Belper et al., 2019) to help find underrepresented views of the complex. With additional data collection and analysis, we feel confident we can obtain a high-resolution structure of this complex and find key domain interactions that we can mutate and test biochemically.