A new tool in RNA research: using an expanded genetic repertoire for site-specific incorporation of functional groups

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分子通常能够采用许多结构，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。这种系统的一个明显的例子是核糖体，在核糖体中，核糖体RNAs和tRNAs驱动化学和构象变化，涉及解码mRNA分子和合成蛋白质。一个更有趣、更广为人知的例子是RNA剪接，在这个例子中，大段RNA从新转录的RNA中移位形成mRNA。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。我们建议通过一种激进的新策略来克服这一点，在该策略中，我们利用了两个新的碱基(表示为Z和P)，这两个碱基可以相互碱基配对，并且已知在DNA合成反应中工作得很好，例如聚合酶链式反应。我们将通过PCR创建RNA转录模板，在模板链的特定位置放置Z和P碱基。我们将制作P和Z的RNA版本，加入化学基团，使我们能够在碱基上添加荧光标记(Z和P的标记不同)。RNA将与标记一起在P和Z处进行修饰，我们将使用双标记的RNA作为底物进行单分子研究的剪接反应。这将对RNA研究产生重大影响，我们将努力确保修饰的碱基可以商业化，并确保轻松跟踪RNA荧光能量转移的能力被认为是打开了新的机会，以寻找影响基于RNA的反应的药物。

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 (thermaly-driven) or as a result of the actions of enzymes. An obvious example of such a system is the ribosome, in which ribosomal RNAs and tRNAs drive chemical and conformational changes involved in decoding a mRNA molecule and synthesizing a protein.A more intriguing and far lass 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 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 a radical new strategy, in which we take advantage of two new bases (representated as Z and P) that can base-pair to each other and are known to work well in DNA synthesis reactions such as PCR. We will create templates for transcription of RNA by PCR in which we place a Z and a P base at specific sites in the template strand. We will make RNA versions of P and Z, incorporating chemical groups that will allow us to add fluorescent labels to the bases (different ones for Z and P). The RNA will be modified at P and Z with the labels, and we will use the doubly-labelled RNA as a substrate in splicing reactions for single molecule studies. This will have a major impact in RNA research, and we will try to ensure both that the modified bases become commercially available and that the ability to follow RNA fluorescence energy transfer easily is recognised as opening up new opportunities to search for drugs that affect RNA-basd reactions.