Department of Biological Sciences, University of Cyprus, P,O, Box 20537, 1678 Nicosia, Cyprus.
J Nanobiotechnology. 2009 Dec 10;7:9. doi: 10.1186/1477-3155-7-9.
We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN). The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.
我们描述了一种基于内含肽的方法，可将量子点（QDs）特异性地连接到体内的靶蛋白上。这种方法允许将任何纳米结构和/或纳米器件共价连接到任何蛋白质上，从而将这种材料靶向到胚胎细胞内的任何细胞内隔室或信号复合物中。我们将一个 PH 结构域与一个分裂内含肽（IN）的 N 端半部分进行基因融合。内含肽的 C 端半部分（IC）在体外与 QD 连接。IC-QD 和编码 PH-IN 的 RNA 被微注射到非洲爪蟾胚胎中。体内内含肽剪接导致完全功能性的 QD-PH 缀合物，可在活体胚胎中实时监测。使用近红外（NIR）发射 QD 可在 EGFP 无法检测到的胚胎深度监测 QD 缀合物，证明了 QD 在这种类型的实验中的优势。总之，我们开发了一种新的体内方法，用于将 QD 和其他人工结构特异性地连接到不同细胞内隔室和信号复合物中的靶蛋白上。
