Structural insights into the effects of 2′-5′ linkages on the RNA duplex

Significance The nonenzymatic replication of RNA is thought to have been a critical step in the emergence of simple cellular life from prebiotic chemistry. However, the chemical copying of RNA templates generates product strands that contain 2′-5′ backbone linkages and normal 3′-5′ linkages. Our recent finding that RNAs with such mixed backbones can still fold into functional structures raised the question of how RNA accommodates the presence of 2′-5′ linkages. Here we use X-ray crystallography and molecular dynamics simulations to reveal how 3′-5′–linked RNA duplexes accommodate interspersed 2′-5′ linkages. The diminished thermal and chemical stability of such RNA duplexes reflects local structural changes, but compensatory changes result in a global RNA duplex structure with relatively minor alterations. The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2′-5′ linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2′-5′ linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2′-linked sugars were in the expected 2′-endo conformation, some were partially or fully in the 3′-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2′-5′ linkages. Our results contribute to the view that a low level of 2′-5′ substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.