Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform. (C) 2012 Elsevier Ltd. All rights reserved.