Development of Near-Infrared Nanoscissor for Down Regulation of Target Genes

• Main Authors: Yen-ChiChiu, 邱彥綺
• Other Authors: Dar-Bin Shieh
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/06218400743614756360

碩士 === 國立成功大學 === 口腔醫學研究所 === 101 === 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.