EVALUATING APTABLOTS AND SOUTHWESTERN- BLOT ASSAYS AS ALTERNATIVES FOR THE SENSITIVE DETECTION OF ALL HISTONES IN TOTAL CELL LYSATES.

MARIÁNGELES CLAUZURE; MARÍA MACARENA MASSIP COPIZ; CRISTIAN ASENSIO; TOMÁS A. SANTA COLOMA

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Abstract: Histones PTMs are markers of epigenetic events and extracellular histones modulate inflammatory, autoimmune and innate immune responses. It is useful to detect histones by sensitive assays. Since they evolved to bind DNA, several biotinylated oligonucleotides and one aptamer were evaluated as reagents for the colorimetric or chemiluminescent detection of endogenous histones bound to nitrocellulose. Many assay variables were systematically adjusted. These south-western or aptablot techniques resulted superior alternatives to western-blot with antibodies. Cell lines or organs were lysed to study the oligo-histones binding profiles resolved by SDS-PAGE. For the TOTAL DETECTION of the 5 histones, biotinylated DNA was an optimal, cost-effective tool, complementary or supplementary of antibodies. We characterized oligonucleotide-histones interactions modulating the components of solutions in the steps of membrane blocking, DNA binding or washing and controlled which histone was detected preferentially adjusting the ionic strength. When needed, histone detection was completely prevented by adding polyanionic reagents (p-value ≤0.01). Testing 1 ssDNA aptamer published as H4 specific but not selected/validated in a lysate context, it reproducibly failed (p-value ≤0.01) to differentiate its H4 target from the other core histones indicating that the selection environment is important in specificity determination. Finally, we developed novel, reliable, non-proteic, coloured MW trackers for better histone resolution in gels. Our procedures showed reproducible analytical advantages and may aid the design of histone bioassay platforms. With a single reagent at picomolar concentration all histones can be sensitively detected for signal normalization in membranes. Our strategies are expected to improve DNA aptamer selection protocols and to facilitate studies of histones for epigenetic, proteomic or immunity research. Keywords: histones, epigenetic, proteomics