Department of Chemistry, Washington University, St. Louis, MO 63130, USA.
Org Biomol Chem. 2013 May 21;11(19):3159-67. doi: 10.1039/c3ob26923j. Epub 2013 Mar 28.
Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA·DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.
通过使用针对 mRNA 的荧光反义探针进行基因表达的光学成像是一个非常感兴趣的领域。开发高灵敏度反义荧光成像剂的主要障碍是探针的细胞内传递效率低下和未结合探针的背景信号高。双反义探针作为 mRNA 成像剂显示出巨大的潜力，因为只有当两个探针同时结合到 mRNA 靶位点时，才会产生信号。由于 RNA 折叠和体内蛋白质结合以及需要结合两个探针，因此选择可及的结合位点变得很困难。更成问题的是，缺乏有效的细胞质内递呈探针的方法，而这些方法最终适用于动物体内的应用。本文报道了使用阳离子壳交联 knedel 样纳米颗粒（cSCK）递呈的 PNA·DNA 双 FRET 探针在活鼠巨噬细胞中 iNOS mRNA 表达的成像。我们首先证明了 PNA 探针和 PNA·DNA 杂交探针都可以在体外转录的 mRNA 上观察到 FRET。然后，我们证明了当杂交探针用 cSCK 转染时，可以在活细胞中观察到 FRET 信号，并且 FRET 信号的强度是序列特异性的，并且取决于 mRNA 的表达水平。
