Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.
Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.
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DOI：
10.1021/ja102585v
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发表时间：
2010-07-21
影响因子：
15
通讯作者：
Wright, David W.
中科院分区：
文献类型：
--
作者：
Jayagopal, Ashwath;Halfpenny, Kristin C.;Perez, Jonas W.;Wright, David W.
提出了一种使用发夹DNA功能化的金纳米颗粒（hAuNP）对信使RNA（mRNA）进行活细胞成像的策略。hAuNP通过其在活细胞内的有效内化而无需转染试剂、改善的对DNA酶降解的抗性、低细胞毒性和发夹DNA分子信标的掺入来改善用于研究RNA运输的技术，从而赋予靶mRNA序列高特异性和灵敏度。此外，靶向的纳米颗粒-信标构建体一旦与靶mRNA序列结合，就保持与靶杂交，从而能够进行RNA运输和下游分析的空间和时间研究。靶向的hAuNP对正常HEp-2细胞中的甘油醛-3-磷酸脱氢酶（GADPH）mRNA和呼吸道合胞病毒（RSV）感染的HEp-2细胞中的RSV mRNA表现出高特异性，具有高的靶与背景比。通过适当选择分子信标荧光团，能够实现活细胞中不同mRNA的多重荧光成像和固定细胞中具有免疫荧光染色蛋白质靶标的mRNA的同时成像。药理学分析表明，hAuNP通过膜-纳米颗粒相互作用在细胞内内化。hAuNP是用于实时分析活细胞中mRNA转运和加工以阐明生物过程和疾病发病机制的有前途的方法。
A strategy is presented for the live cell imaging of messenger RNA (mRNA) using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNAse degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells, and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently-stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.
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