# 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.

## Key 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.