Arginine Cofactors on the Polymerase Ribozyme

The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor

The structural basis of RNA-catalyzed RNA polymerization

Early life presumably required polymerase ribozymes capable of replicating RNA. Known polymerase ribozymes best approximating such replicases use as their catalytic engine an RNA-ligase ribozyme originally selected from random RNA sequences. Here we report 3.15-Å crystal structures of this ligase trapped in catalytically viable preligation states, with the 3′-hydroxyl nucleophile positioned for in-line attack on the 5′-triphosphate. Guided by metal- and solvent-mediated interactions, the 5′-triphosphate hooks into the major groove of the adjoining RNA duplex in an unanticipated conformation. Two phosphates and the nucleophile jointly coordinate an active-site metal ion. Atomic mutagenesis experiments demonstrate that active-site nucleobase and hydroxyl groups also participate directly in catalysis, collectively playing a role that in proteinaceous polymerases is performed by a second metal ion. Thus artificial ribozymes can use complex catalytic strategies that differ markedly from those of analogous biological enzymes.National Institutes of Health (U.S.) (grant GM06183)National Center for Research Resources (U.S.) (award RR-1530)United States. Dept. of Energy (Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH1135

An RNA toolbox for cancer immunotherapy

Cancer immunotherapy has revolutionized oncology practice. However, current protein and cell therapy tools used in cancer immunotherapy are far from perfect, and there is room for improvement regarding their efficacy and safety. RNA-based structures have diverse functions, ranging from gene expression and gene regulation to pro-inflammatory effects and the ability to specifically bind different molecules. These functions make them versatile tools that may advance cancer vaccines and immunomodulation, surpassing existing approaches. These technologies should not be considered as competitors of current immunotherapies but as partners in synergistic combinations and as a clear opportunity to reach more efficient and personalized results. RNA and RNA derivatives can be exploited therapeutically as a platform to encode protein sequences, provide innate pro-inflammatory signals to the immune system (such as those denoting viral infection), control the expression of other RNAs (including key immunosuppressive factors) post-transcriptionally and conform structural scaffoldings binding proteins that control immune cells by modifying their function. Nascent RNA immunotherapeutics include RNA vaccines encoding cancer neoantigens, mRNAs encoding immunomodulatory factors, viral RNA analogues, interference RNAs and protein-binding RNA aptamers. These approaches are already in early clinical development with promising safety and efficacy results