Repurposing the Escherichia coli HU as a Universal dsDNA Amplicon Detection Tool

Identificador:  imarina:9633000

Handle: https://hdl.handle.net/20.500.11797/imarina9633000

Autors:  Ruhela A; Choudhary M; Skouridou V; Masip L

Resum:
DNA binding proteins interact with DNA in a sequence-specific manner or indiscriminately without sequence specificity. While these properties make them highly attractive for assay development, their use as DNA biorecognition elements has remained limited. In this work, we describe a new application using the non-sequence specific Escherichia coli histone-like HU protein for general DNA detection. We engineered a single-chain HU heterodimer (scHupBA) that exhibits high binding affinity for double-stranded DNA (≥50 bp) and considerably lower affinity for single-stranded DNA. By combining this protein with a scHupBA-horseradish peroxidase conjugate, we developed an assay capable of detecting PCR amplicons, either post-purification or directly within a PCR reaction, using microtiter plates and magnetic beads. This system efficiently detected DNA fragments >100 bp regardless of sequence, with detection signals increasing non-linearly with fragment length. To the best of our knowledge, this is the first report of the use of a non-sequence-specific double-stranded DNA binding protein for general DNA detection. The approach described herein is target-independent, it does not require any primer modification or labeling nor the use of expensive reagents. It is an overall cost-effective and efficient method for universal dsDNA amplicon detection, especially for rapid analysis of amplification reactions.