6 Crystal structure studies of RNA molecules containing 2′–5′-linkages

Abstract

RNA can play dual roles as a carrier of genetic information and as a catalyst of specific reactions, and it may have been the first biopolymer to have emerged on the early earth. The non-enzymatic replication of RNA was likely a key step in the evolution of simple cellular life from prebiotic chemistry. In the current model of template-directed polymerization of activated monomers, the chemical copying of RNA always generates a mixture of 3′–5′ and 2′–5′ backbone linkages due to the similar nucleophilicity and orientation of the 2′ and 3′ hydroxyl groups on the ribose. This lack of regiospecificity has been regarded as a central problem for the evolution of functional RNAs, since the resulting backbone heterogeneity was expected to disrupt their folding, molecular recognition and catalytic properties of functional RNAs such as ribozymes. However, a recent study from our lab has demonstrated that RNAs with a certain percentage of 2′–5′ linkages can still retain RNA functions, for example, in a FMN-binding aptamer and a hammerhead ribozyme system. More interestingly, it has been known for a long time that 2′–5′ linkages can reduce the melting temperature of RNA duplexes, making it easier to separate the strands. Although the detailed mechanism is still not clear, considering that strand separation is another unsolved big problem for non-enzymatic RNA replication, this feature may actually afford a selective advantage to duplexes exhibiting backbone heterogeneity. In addition, previous studies have revealed that 2′–5′ linkages in a RNA duplex are more easily hydrolyzed compared to normal 3′–5′ linkages. Thus, there is a selective advantage for the evolution of homogeneous RNA systems with more accurate replication. Altogether, the coexistence of 2′–5′ and 3′–5′ linkages may be a central feature that allowed RNA to play a central role in the original stage of life. In this work, we will present several X-ray crystal structures of RNA duplexes and an aptamer that contain 2′–5′ linkages. These structures help us to understand how RNA can adjust its structure to accommodate the backbone heterogeneity.