FRET from Quantum Dots to Photodecompose Undesired Acceptors and Report the Condensation and Decondensation of Plasmid DNA

Abstract

Protection of genes against enzymatic degradation and overcoming of cellular barriers are critical for efficient gene delivery. The effectiveness of gene delivery by nonviral vectors depends mostly on the extent of DNA packaging or condensation. We show that Förster resonance energy transfer (FRET)-mediated photodecomposition of undesired acceptors in doubly labeled plasmid DNA (pDNA) and FRET recovery after acceptor photodecomposition (FRET-RAP) are effective methods for the detection of DNA condensation and decondensation. Our hypothesis is that undesired acceptors within the Förster distance of highly-photostable donors in precondensed DNA can be selectively photodecomposed by FRET. We investigate this hypothesis by the random labeling of pcDNA3.1-GL3 and pUC18DNA with quantum dots (QDs) as the energy donor and AlexaFluor594 or Cy5 as the acceptor. At first, the random labeling generates efficient FRET, also called intrinsic FRET, in precondensed DNA, which prevents us from decoding any changes in the FRET efficiency during DNA condensation. Next, we suppressed the intrinsic FRET by the FRET-mediated photodecomposition of acceptors within the Förster distance of QDs. Conversely, many acceptors kept intact beyond the Förster distance provide us with high FRET efficiency during the condensation of pDNA using protamine. Further, the FRET efficiency is significantly decreased during the decondensation of DNA using heparan sulfate and glutathione. The random labeling of DNA using excess acceptors around photostable donors followed by the FRET-mediated photodecomposition of undesired acceptors can be a promising method for not only the sensitive detection of DNA condensation by FRET but also the customization of biomolecular sensors.