RNA conformation and metal ion coordination in the nonenzymatic primer extension reaction center

Abstract

ABSTRACTNonenzymatic template-directed RNA primer extension with 2-aminoimidazole activated nucleotides begins with the generation of an imidazolium-bridged dinucleotide intermediate that binds to the template adjacent to the primer. Crystal structures have illuminated the overall conformation of the primer/template/bridged-dinucleotide complex, but critical aspects of the structure remain unseen or unresolved. Most significantly the catalytic metal ion has not been visualized, and its coordination to functional groups in the reaction center remains unknown. In addition, the orientation of the imidazolium moiety of the bridged dinucleotide is unresolved, and static crystal structures do not provide insight into the dynamic aspects of catalysis. To address these questions, we performed atomistic molecular dynamics simulations of primer/template/bridged-dinucleotide/helper-oligonucleotide complexes. Our simulations suggest that one particular orientation of the imidazolium moiety of the bridged dinucleotide is likely to be more favorable for the reaction. Additionally, Mg2+ions are known to play an important role in the catalysis of RNA copying chemistry. We have examined potential Mg2+contacts with multiple oxygen atoms in the reaction center. Our simulations suggest a preferred coordination of the catalytic Mg2+between O3′ of the terminal primer nucleotide and one of the non-bridging oxygens of the reactive phosphate of the imidazolium-bridged dinucleotide. We also observe significant stabilization of the geometry of the reaction center in this preferred coordination of the catalytic Mg2+. Our simulations suggest that the catalytic metal ion plays an important role in overcoming electrostatic repulsion between a deprotonated O3′ and the incoming phosphate of the bridged dinucleotide.