Antibody-Free Reading of the Histone Code Using a Simple Chemical Sensor Array

Abstract

The histone code refers to the complex network of histone post-translational modifications that control gene expression and are of high interest as drivers of a large number of human diseases. We report here on a mix-and-match toolkit of readily available dyes and calixarene host molecules that can be combined to form dye-displacement sensors that respond to a wide variety of cationic peptides. Using the data from only two or three such simple supramolecular sensors as a chemical sensor array produces fingerprints of data that discriminate robustly among many kinds of histone code elements. "Reads" that are accomplished include the discrimination of unmethylated, mono-, di-, and trimethylated lysines on a single histone tail sequence, identification of different modifications and combinations of modifications on a single histone tail sequence, identification of a single modification type in several different sequence contexts, and identification of isomeric dimethylarginine modifications. Reads that are sometimes troublesome for antibodies are achieved. We also report on the ability of the sensor array to report simultaneously on the concentrations and identities of histone modifications. This sensor array discriminates between post-translationally modified analytes without being limited to partners that contain a single, programmed binding interaction.

Metal complexes as “protein surface mimetics”

“…31 Please do not adjust margins Please do not adjust margins assays/sensors for antibody free detection of histone modifications through lysine side chain recognition. 35,36 …”

Dynamically Analyte-Responsive Macrocyclic Host–Fluorophore Systems

“…The underlying idea was that− although ACh and Ch could not be differentiated on account of their identical positive charge and the same NMe 3 + recognition motif, the oxidation product of Ch, betaine, could be detected, because it is zwitterionic, and therefore much more weakly bound ( Figure 5, right). LCG (Figure 2b) was found to be an excellent indicator dye for CX4 (fluorescence enhancement 22 Thus, when the reaction was conducted in the presence of an excess of choline oxidase and a ratedetermining concentration of acetylcholinesterase, the CX4· LCG reporter pair afforded a decrease in fluorescence signal. This resulted from the formation of betaine as weak competitor, which allowed the dye to be taken up by the macrocycle in the course of the reaction.…”

Synthetic trimethyllysine receptors that bind histone 3, trimethyllysine 27 (H3K27me3) and disrupt its interaction with the epigenetic reader protein CBX7

“…Previous studies have shown that the dye lucigenin is a promiscuous binder of many different sulfonated calixarenes, 39,40 and we found it to be generally well suited to use in a dye-displacement format with each of these compounds. We sought to create a rapid, quantitative assay for H3K27me3 (and H3K27) binding according to the schematic in Fig.…”