Detecting cytosine methylation at the single DNA molecule level

虽然许多生物(人类、植物、无脊椎动物和脊椎动物)的基因组已经被测序，并识别了许多基因，但由于缺乏分析技术，我们对这些基因的调控的了解是有限的。DNA由四个核酸碱基组成，分别是腺嘌呤、鸟嘌呤、胞嘧啶和胸腺嘧啶。其中一些核酸碱基可以被酶修饰，结果是增加了一个甲基；在这里，我们将研究新的技术来检测单个DNA分子中的甲基胞嘧啶和未甲基化胞嘧啶。胞嘧啶核酸碱基的甲基化与基因沉默有关。在人类中，DNA甲基化被认为在发育中起着关键作用，在许多疾病中都是异常的，但到目前为止，其完整的作用尚不清楚。检测DNA中甲基化胞嘧啶的技术有很多，但目前的方法还没有提供一种简单、快速、可靠、廉价的方法。一个主要问题是需要评估细胞样本的DNA，这些细胞样本将包含相同的DNA序列，但相对于甲基化的胞嘧啶残基是不同的。因此，通常会得到一个平均值。那些确实允许对单DNA链进行评估的技术是非常费力和有限的。在这里，我们将开发一种新的方法，在单分子水平上检测含有甲基化胞嘧啶的序列。目前还有其他小组在单分子DNA测序领域工作，但这些方法速度很慢，而且DNA是作为单链进行研究的。我们将询问双链DNA，这将使我们能够检测到每条链上甲基化或未甲基化的胞嘧啶分子，称为半甲基化。我们的方法是创造一种人工形式的DNA，一种寡核苷酸，它以特定的序列结合并包裹在双链DNA分子的主槽中。这种人造形式的DNA当结合在一起时被称为三链，合成的分子也将包含一个荧光团。当DNA样本被这些形成三链的寡核苷酸处理后，螺旋将在其沿途的不同点包含荧光团。我们将DNA样本注入到一个小通道中，这将导致链的解开和拉直，这样它就会被穿入光学询问通道。荧光团将被光激发，在纳米通道内存在纳米结构时，将导致荧光强度的变化。强度的变化将提供一个代码，表明不同的胞嘧啶序列(未甲基化、半甲基化、双甲基化)的甲基化状态。一种检测DNA序列中甲基化和非甲基化胞嘧啶的简单技术对于广泛的学术、临床和工业研究社区将是重要的，因为这将使人们更好地了解基因调控。在许多研究领域中，胞嘧啶甲基化被认为在人类中起着重要作用，如饮食相关疾病、炎症性疾病、胚胎发育等，或者在植物中被认为是了解环境胁迫的影响。但如上所述，胞嘧啶甲基化对许多生物体来说很重要，而一种允许分析基因内甲基化模式的技术从长远来看具有潜在的商业价值。首先，需要更好地了解DNA甲基化，但这里提出的某种形式的方法可能会产生一种诊断工具。

Although the genomes of many organisms (humans, plants, invertebrates and vertebrates) have been sequenced and many of the genes identified, our understanding of the regulation of the genes is limited due to lack of analysis technology. DNA is composed of four nucleic acid bases, adenine, guanine, cytosine and thymine. Some of these nucleic acid bases can be modified by enzymes and as a result have an additional methyl group; here we will investigate new technologies for the detection of methylcytosine and unmethylated cytosine within single DNA molecules. The methylation of cytosine nucleic acid bases is associated with gene silencing. In humans DNA methylation is considered to play a critical role in development and is aberrant in many diseases, but as yet the complete role remains unclear. There are numerous techniques for the detection of methylated cytosine in DNA, but the current methodologies do not yet provide a simple, fast, reliable cheap approach. A major problem is the need to evaluate DNA from cell samples that will contain the same DNA sequence but which are heterogeneous with respect to the cytosine residues that are methylated. So an average is often obtained. Those techniques that do allow single DNA strands to be evaluated are highly laborious and limited. Here we will develop a new approach for detecting sequences containing methylated cytosines at the single molecule level. There are currently other groups working in the field of DNA sequencing of single molecules, but these methods are slow and the DNA is investigated as a single strand. We will interrogate double-stranded DNA and this will allow us to detect methylated or unmethylated cytosine molecules on each strand, called hemi-methylation. Our approach is to create an artificial form of DNA, an oligonucleotide, that associates and wraps within the major groove of double-stranded DNA molecule at specific sequences. This artificial form of DNA when associated is called a triplex and the molecules synthesised will also contain a fluorophore. When the DNA sample has been treated with these triplex forming oligonucleotides the helix will contain fluorophores at different points along it. We will inject the DNA sample into a small channel that will result in unravelling and straightening of the strand so that it is then threaded into an optical interrogation channel. The fluorophores will be excited with light, which in the presence of nanostructures within the nanochannel will result in fluorescence intensity changes. The change in intensity will provide a code that indicates the methylation status of the different cytosine containing sequences (unmethylated, hemi-methylated, doubly methylated). A simple technique to detect the methylated and unmethylated cytosines within DNA sequences will be important for a wide academic, clinical and industrial research community, since this will allow a greater understanding of gene regulation. There are many research areas where cytosine methylation is considered to play a significant role in humans, such as diet related disease, inflammatory diseases, embryonic development to name a few, or in plants for understanding the effect of environmental stress. But as noted above, cytosine methylation is important for many organisms, and a technique that allows for the analysis of the patterns of methylation within genes has the potential to be commercially valuable in the longer-term. First a better understanding of DNA methylation is required, but it is possible that a form of the approach proposed here will yield a diagnostic tool.