已發表之人造標靶性光激發奈米剪技術 (Artificial, Targeted, Light-Activated Nanoscissor , ATLANS) 可利用特定波長光子激發切割試劑產生自由基而剪切標靶基因。然而其激發波長 (λ=460 nm) 無法穿透深層組織，且可能造成非標靶基因非特異性傷害。本研究發展近紅外光奈米剪 (Near-Infrared, Artificial, Targeted, Light-Activated Nanoscissor, NIR-ATLANS)將激發波長改為808 nm，同時利用髮夾結構與光淬設計促進切割專一性。此NIR-ATLANS以金奈米棒 (gold nanorod, Au NR) 為核心載體以提供近紅外光區段之表面電漿共振淬火 (quench)功能及搭載終端修飾近紅外光切割試劑 多次甲基菁 (cypate) 的三股寡核苷酸 (triplex-forming oligonucleotides, TFOs)的平台。此單股核苷酸可與標靶基因形成專一性的三股結構並將原來結構上受到淬火抑制之切割分子轉為準活化態以利後續標靶切割之進行。此一設計降低了非標靶性切割機率而保護DNA免於受損。當NIR-ATLANS-標靶DNA複合結構受到近紅外光激活時，cypate能釋放自由基而切割DNA。我們在NIR-ATLANS表面合成PEG修飾(PEGylated NIR-ATLANS)而顯著提增其生物相容性。凝膠遷移實驗證實篩選出之TFO能與目標雙股DNA專一性結合。一金奈米棒則約可修飾180個TFO。共聚焦顯微鏡觀察證實PEGylated NIR-ATLANS能於處理細胞12小時後進入細胞核。而以穩定表現EGFP之細胞株為模式，給予PEGylated NIR-ATLANS並照射808 nm 雷射後，可由流式細胞儀定量分析發現細胞EGFP螢光於48小時達到最佳抑制效果。反之，控制組別 (scrambled control)細胞則沒有顯著EGFP表現量下降的現象。期望未來PEGylated NIR-ATLANS能提供癌症基因治療之一新穎方向。

We previously developed an artificial, targeted, light-activated nanoscissor (ATLANS) for precise photonic cleavage of target gene sequence. This 1st generation ATLANS was designed to be activated by blue light (λ=460 nm) thus limited by photon penetration depth in tissues. This study aims to develop a new generation ATLANS activated by near-infrared (808 nm). The NIR-ATLANS comprised of gold nanorod (Au NR) core with a monolayer of cypate-modified triplex-forming oligonucleotides (TFOs) that recognize targeted DNA duplex. The Au NR acts as a quencher to prevent the excitation energy from off-targeting activation thus to protect the cell from non-specific DNA damage. The beacon TFOs was end-modified with cypate, an ICG (indocyanine green) analogue, to form a hairpin structure that only capable of being activated when extended away from the Au NR surface plasma quench upon recognition of the target gene sequence. After laser exposure, the NIR-ATLANS generates free radical from cypate and induced target DNA break. Modification of PEG to the surface of NIR-ATLANS (PEGylated NIR-ATLANS) significantly improved biocompatibility. The electrophoretic mobility shift assay showed selective binding of TFOs to the target EGFP sequence. We demonstrated a maximum coverage up to 180 TFOs per Au NR. Further, we discovered through confocal microscopy that PEGylated NIR-ATLNAS could shuffle to the nucleus directly in 12 hours after cellular uptake. When activated by 808 nm laser, the PEGylated NIR-ATLANS down regulate target gene EGFP that plateau at 48 hours after lasing compared to the scrambled control group. Such technology holds a great potential in the future cancer gene therapy and genetic engineering.

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