Talk abstracts
Talk on Saturday 12:35-12:55pm submitted by Scott Aoki
Structural biology paired with in silico screening to predict RNA modification-protein interactions
Murphy Angelo (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana ), Wen Zhang (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana ), Jonah Z. Vilseck (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana ), Scott T. Aoki (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana )
Abstract:
RNA modifications shape gene expression through a smorgasbord of chemical changes to canonical RNA bases. Although numbering in the hundreds [1], only a few RNA modifications have been well characterized, in part due to the absence of methods to identify modification sites and how they interact with binding proteins. In silico Multisite Lambda Dynamics is an efficient way to measure free energy differences after changing chemical moieties on molecules [2, 3]. The calculations are computationally efficient and can be tested at multiple sites, enabling full sets of perturbations to be tested in weeks that takes other calculations months. We wanted to utilize Multisite Lambda Dynamics to predict how RNA modifications affect protein binding, and antibodies remain a common tool to identify modified RNA. While straightforward in use, antibody specificity issues can result in off-target binding and confound conclusions [4]. Here, we test Multisite Lambda Dynamics as a computational tool to gauge the specificity of a modification-targeting antibody against a wide spectrum of RNA modifications. We determined the crystal structures of an Inosine and an N6-methyladenosine (m6A) targeting antibody fragment in complex with their modified RNA base to 2.0 Å and 2.65 Å, respectively. Using these structures as starting points, we utilized Multisite Lambda Dynamics to screen whether 42 different bases could potentially bind. The simulations predicted RNA modifications that permit or inhibit binding to antibodies, and in vitro binding assays supported these results. In sum, Multisite Lambda Dynamics may be used as a preliminary screen to predict antibody specificity against a large library of RNA modifications. More importantly, this strategy promises to be an innovative way to elucidate how the hundreds of known RNA modifications interact with proteins and other biological molecules to elucidate their mechanisms in gene expression.
References:
1. McCown, P.J., et al., Naturally occurring modified ribonucleosides. Wiley Interdiscip Rev RNA, 2020. 11(5): p. e1595.
2. Vilseck, J.Z., et al., Predicting Binding Free Energies in a Large Combinatorial Chemical Space Using Multisite λ Dynamics. The Journal of Physical Chemistry Letters, 2018. 9(12): p. 3328-3332.
3. Knight, J.L. and C.L. Brooks, 3rd, Multi-Site lambda-dynamics for simulated Structure-Activity Relationship studies. J Chem Theory Comput, 2011. 7(9): p. 2728-2739.
4. Grozhik, A.V., et al., Antibody cross-reactivity accounts for widespread appearance of m(1)A in 5'UTRs. Nat Commun, 2019. 10(1): p. 5126.
Keywords: RNA modifications, antibody, computational modeling