Technology for Synthesis of Chemically Diverse RNAs

Summary RNA synthesis technologies have become critical in basic biomedical research and in development of RNA based sensors diagnostics and therapeutics Currently RNAs are synthesized by either solid phase phosphoramidite chemistry or by transcription in solution with phage encoded RNA polymerases The structural diversity of chemically synthesized RNAs is limited by the availability of the phosphoramidites and the compatibility of reaction chemistry with different nucleotide modifications but the more important limitation of chemical synthesis is that RNAs longer than a few tens of nucleotides cannot be made with acceptable yields Enzymatic methods allow synthesis of much longer RNAs and by using specially engineered RNAPs even allow synthesis of RNAs with non canonical chemistry to enhance functionality or RNAse resistance but a limitation of enzymatic synthesis is that the chemical composition of an RNA is homogeneously determined by the mix of NTPs in the reaction i e different segments of one RNA cannot have different chemistry We recently published a proof of principle study for a new RNA synthesis technology PLOR for positional labeling of RNA that combines solid phase and enzymatic synthesis to allow preparation of indefinitely long RNAs in which multiple specific segments or nucleotides can be specifically labeled with distinct chemistry This technology represents a quantum leap in our ability to characterize RNA structure and mechanism to prepare RNA aptamers or interfering RNAs specifically derivatized to optimize their delivery stability or effectiveness in vivo and also presents potential advantages in efficiency that may supersede current synthesis methods even for conventional single chemistry RNAs The proposed work will optimize the efficiency and economics of this novel technology expand its utility for synthesis of RNAs with non canonical nucleotide chemistries and in general make possible the routine and economically efficient preparation of a new generation of mosaic chemically diverse RNA molecules with applications ranging from fundamental biomedical research to clinical therapeutics Awareness of the importance of naturally occurring RNAs in normal and diseased cell function has grown enormously over the past two decades and the applications of synthetic RNAs in research diagnostic and synthetic applications has also exploded during this period Both studies of natural RNAs and the effectiveness of synthetic RNAs would be enhanced by technologies allowing more precise control of the chemistry of synthetic RNAs The technology to be developed in this work will make this possible and will have a large impact on this growing field of biomedicine