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Drugs which target ribonucleic acids (RNA) make incremental progress in the clinic. This is to be expected for new classes of compounds targeting untested classes of macromolecular targets which cannot be addressed by the usual small molecule drugs. In this context, therapeutic oligonucleotides (oligomers composed of 15-20 nucleotide units), an experimental class of drug molecule, have a tremendous potential in medicine, yet are not pursued by the mainstream pharmaceutical industry because of the outstanding technical challenges associated with developing large and complex drug molecules. The recent discovery of a new class of natural small RNAs (microRNAs or miRNAs) which contribute to a number of mechanisms of disease represents a new class of RNA target for drugs and has fueled motivation to overcome these challenges, especially in the academia and biotech sectors. One particular miRNA, mir-122, a human miRNA expressed in the liver which is used by the hepatitis C viral RNA during viral replication, is the target for an oligonucleotide (miravirsen) in late-phase clinical trials. The research plan in this application represents a proposed extension of funded work currently ongoing which expires at the end of 2012, the principal objective of which is generally to make oligonucleotide drugs more tractable.Current dogma states that oligonucleotide drugs need to be large in order to have high binding affinity and high selectivity for their targets in cells. In the current project we are attempting to decrease the length of oligonucleotide drugs indirectly by identifying optimal binding sites on the target (pre-mir-122: a precursor of mir-122) which are structurally pre-disposed such that oligonucleotide drugs bind with high affinity. We hypothesized that such sites are present amongst the three-dimensional structures of pre-miRNAs, and we have established novel methods (in vitro and cellular assays) to identify these sites during the current project. Targeting miRNA precursors is a novel high-risk approach rarely pursued by other groups. To date, we have succeeded to show using in vitro enzymatic assays and cellular assays that oligonucleotides composed of “standard” ribonucleotides targeting these “favored” sites can inhibit their target. However, they only show low efficiency in cells. In this proposed extension of the project we propose to build on this foundation with a new approach to directly increase the binding affinity of the oligonucleotides. We believe that this will in turn increase efficiency in cellular assays. This comprises the design, synthesis and incorporation of non-natural nucleotide building blocks into the drugs. Briefly, we will append small functional groups to certain nucleotides of the oligonucleotide which will endow the drug with additional binding affinity without adding unduly to their size. We estimate to be able to synthesize and test approximately 100-200 new molecules against mir-122 in dedicated assays which we developed during the currently funded project. The fragments will be attached to the nucleotides such that they protrude into two types of binding pocket or “groove” on the target pre-mir-122 and increase the binding affinity of the overall interaction, for example electrostatically. Fragments will be selected from those which have previously been reported in literature to bind to RNA structures. We will determine the source of increased (or decreased) binding affinities with respect to the rates of association and dissociation (kon and koff, respectively) of the molecules with the target using surface plasmon resonance. The investigation will demonstrate the suitability of the “major groove” and the “minor groove” in general as a favored binding site for small molecule fragments providing guidance for further unrelated projects with other miRNAs or alternative classes of the numerous RNA targets in cells. The research plan is designed for two PhD students however the content of the proposal contains numerous possibilities for additional research projects.