An electrophoretic mobility shift assay with chemiluminescent readout to evaluate DNA-targeting oligonucleotide-based probes

TL;DR

This paper introduces a simple method to test how well DNA-targeting probes bind to DNA using electrophoresis and light-based detection.

Contribution

A new, accessible protocol combining electrophoretic mobility shift and chemiluminescent detection for evaluating DNA probe binding.

Findings

The method effectively evaluates dsDNA recognition by various probes.

LNAs, Invader probes, and chimeric probes show different dsDNA-binding efficiencies.

R-based scripts enable data visualization and C50 value determination.

Abstract

A comprehensive and user-friendly method for evaluating recognition of double-stranded DNA (dsDNA) targets by oligonucleotide-based probes is presented. Thus, dsDNA-targeting probes such as single-stranded locked nucleic acids (LNAs) and double-stranded Invader probes are incubated with digoxigenin-labeled DNA hairpin targets, and the resulting recognition complexes are resolved using an electrophoretic mobility shift assay and tagged using a chemiluminescence immunoassay. Emissive products are detected by a C-DiGit Blot Scanner and quantified with the accompanying software. R-based scripts for data visualization and determination of C50 values (a measure of the dsDNA-binding affinity of a probe) are also provided. The data presented here demonstrate the effectiveness of the described protocol and highlight the variable dsDNA-recognition efficiencies of LNAs, Invader probes, and chimeric Invader:LNA probes.

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