A new genetically-encoded aptamer platform for multi-colour RNA imaging

哺乳动物细胞中的许多重要过程都涉及到RNA。特别令人感兴趣的是其中RNA分子本身作用于催化影响第二个RNA分子的事件。RNA分子通常能够采用多种结构，它们可以自发地(热驱动)或由于酶的作用而在这些结构之间波动。一个不太为人所知的例子是RNA剪接，在这种剪接中，大段的RNA从新转录的RNA中置换出来，形成信使核糖核酸。剪接机制是以RNA为基础的，RNA底物非常长，位点很难识别，这些位点的使用往往受到复杂的组织特异性调控，可能涉及与RNA形成结构。监测RNA的结构或构象是否发生变化的一个好方法是在RNA的两个位置特别放置荧光标记。这些标记物的选择是这样的，当它们接近时，它们将荧光激发的能量从一个转移到另一个；这是可以测量的。这是跟踪单个分子上的事件的一种特别好的方法，这是研究剪接的基本方法。目前的主要缺点是很难在一个长RNA分子内部的特定位置引入两个标记。我们建议通过遗传编码RNA结构来结合到荧光标签来克服这一点。有了标记RNA的双色系统，将为RNA研究提供一个强大的新工具，因为它将允许直接比较各种RNA处理事件，而不是依赖于单个荧光团的荧光发射。我们的跨学科方法是利用一种名为SELEX的人工进化技术来识别与荧光团结合并显示红色发射的RNA结构(适配子)。然后，我们将把这些适配子结合到长RNA分子中，并研究它们作为RNA生物学报告者的潜力。这将对RNA研究产生重大影响，我们将确保适配子商业化，并确保跟踪RNA荧光的能力被认为打开了寻找影响基于RNA的反应的药物的新机会。

Many important processes in mammalian cells involve RNA. Of particular interest are those in which RNA molecules themselves act to catalyse events that affect a second RNA molecule. RNA molecules are often able to adopt a number of structures, and they can fluctuate between these either spontaneously (thermally-driven) or as a result of the actions of enzymes. A less well understood example is RNA splicing, in which large stretches of RNA are displaced from newly-transcribed RNA to form mRNA. The splicing machinery is RNA-based, and the RNA substrates are very long, sites are hard to recognise, and the use of these sites is often subject to complex tissue-specific regulation that may involve the formation of structures with the RNA. A good way of monitoring whether RNA undergoes changes in its structures or conformations is to specifically place fluorescent labels at two sites in the RNA. These labels are chosen such that, when they come into close proximity, they transfer the energy of fluorescence excitation from one to the other; this can be measured. This is a particularly good method for following the events on a single molecule, which is an essential approach for studying splicing. The main drawback at present is that it is very difficult to introduce two labels at specific sites far inside a long RNA molecule. We propose to overcome this by genetically encoding RNA structures to bind to fluorescent tags. Having available a two-colour system to label RNA will provide a powerful new tool for RNA research as it will allow various RNA processing events to be directly compared rather than relying on fluorescence emission of a single fluorophore. Our inter-disciplinary approach is to exploit an artificial evolution technique known as SELEX to identify RNA structures (aptamers) that bind fluorophores that exhibit red emission. We will then incorporate these aptamers into long RNA molecules and investigate their potential as reporters of RNA biology. This will have a major impact in RNA research, and we will ensure both that the aptamers become commercially available and that the ability to follow RNA fluorescence is recognised as opening up new opportunities to search for drugs that affect RNA-based reactions.