Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously.

中文翻译：

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通过两步 QD-FRET 同时非侵入性分析 DNA 凝聚和稳定性

由可将 DNA 凝聚形成纳米复合物的阳离子聚合物组成的纳米级载体是基因转移的有前途的选择。只有对关键限速步骤（例如纳米复合物解包以释放 DNA 和核酸酶降解）有更好的机械理解，才能合理设计更有效的非病毒基因载体。我们提出了一种两步量子点荧光共振能量转移（两步 QD-FRET）方法来同时且非侵入性地分析 DNA 凝聚和稳定性。用 QD（525 nm 发射）和核酸染料双重标记的质粒 DNA 与 Cy5 标记的阳离子基因载体复合。QD 供体驱动能量逐步通过中间核酸染料转移到最终受体 Cy5。通过能量转移效率的定量比率分析，以单粒子方式和细胞内至少区分出三种不同的 DNA 凝聚和完整性状态。这种新颖的两步 QD-FRET 方法允许更详细地评估同时发生的 DNA 释放和降解。