Next Generation Detection Reagents for Chromatin Immunoprecipitation

PROJECT SUMMARY Understanding the genome wide distribution and organization of various epigenetic marks in the context of disease progression or in response to therapeutic intervention has broad applications for both basic and translational research EpiCypher has developed the first quantitative ChIP approach R HG which uses post translationally modified PTM defined recombinant nucleosomes as internally calibrated spike in standards ICe ChIP This will enable a marked expansion of ChIP applications into new markets including drug discovery and diagnostics though its full potential is restrained by two current limitations in detection reagents First many so called ChIP grade antibodies exhibit extensive cross reactivity with structurally similar PTMs e g trimethylation vs dimethylation at H K H K me vs H K me Second antibody binding is highly influenced by the presence of neighboring PTMs Antibodies targeting H K me for instance exhibit nearly fold greater binding affinity if phosphorylation is also present at nearby residues These specificity limitations result from the crude selection strategies by which antibodies are developed It is therefore imperative to develop next generation detection reagents that can have their binding preferences selected for or tuned using rigorous and efficient selection strategies Here EpiCypher will develop RNA aptamers as highly specific detection reagents for ChIP applications Aptamers are synthetically derived using an in vitro directed evolution approach SELEX Systematic Evolution of Ligands by EXponential enrichment thereby mitigating animal involvement and allowing specific binding preferences to be positively and negatively selected a priori The ability to finely tune aptamer binding affinity and specificity without the expenditure of animal life provides a marked advantage over antibody based reagents Recent advances in aptamer technology have enabled these reagents to be highly resistant to degradation in cell lysates a feature required for ChIP applications However their development towards histone PTMs has been restricted by the highly charged nature of chromatin To meet this challenge EpiCypher is collaborating with Dr Matthew Levy to develop a novel engineering strategy to generate aptamers that can tolerate such highly charged target motifs The innovation of this project is a unique selection strategy and the incorporation of hydrophobic residues to generate aptamers that can bind PTMs on charged histones with exquisite target specificity In Aim we will establish an optimization pipeline to develop aptamers specific for histone H K methylation and validate their binding specificity using EpiCypherandapos s combinatorially modified histone peptide array EpiTitanTM In Aim we will demonstrate the utility of our novel aptamers in ICe ChAP Chromatin AptamerPrecipitation and compare these results with best in class antibodies capable of distinguishing various H K methylation states In Phase II we will scale up commercial production of aptamers that target H K methylation and other disease relevant PTMs PROJECT NARRATIVE EpiCypher is about to commercially release ICe ChIP the first quantitative Chromatin ImmunoPrecipitation approach This disruptive technology will play a key role in advancing the development of epigenetic therapeutics and diagnostics Despite this breakthrough ChIP is ultimately dependent on the use of antibody based detection reagents which have significant limitations in their binding specificity for certain histone post translational modifications Therefore next generation detection reagents are direly needed for advanced ChIP applications Here EpiCypher will develop a novel engineering pipeline to generate RNA aptamers as highly specific detection tools for ChIP applications These aptamers will mitigate current limitations in antibody specificity thereby bringing a greatly needed and novel class of epigenetic reagent to market