Detecting cytosine methylation at the single DNA molecule level

在单个 DNA 分子水平检测胞嘧啶甲基化

• 批准号: BB/I022686/1
• 负责人: Jeremy Baumberg
• 金额: $ 7.18万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI022686%2F1
• 关键词: Detecting; cytosine; methylation; single; DNA

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.

虽然许多生物体（人类、植物、无脊椎动物和脊椎动物）的基因组已经测序，许多基因已经被鉴定，但由于缺乏分析技术，我们对基因调控的理解是有限的。DNA由四种核酸碱基组成，即腺嘌呤、鸟嘌呤、胞嘧啶和胸腺嘧啶。这些核酸碱基中的一些可以被酶修饰，因此具有额外的甲基;在这里，我们将研究用于检测单个DNA分子内的甲基胞嘧啶和未甲基化胞嘧啶的新技术。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关键作用，并且在许多疾病中是异常的，但到目前为止，完整的作用仍然不清楚。DNA甲基化胞嘧啶的检测技术有很多种，但目前的方法学还没有提供一种简单、快速、可靠、廉价的方法。一个主要的问题是需要评估来自细胞样品的DNA，所述细胞样品将含有相同的DNA序列，但其相对于甲基化的胞嘧啶残基是异质的。因此，通常会得到平均值。那些确实允许评估单链DNA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来检测序列含有甲基化胞嘧啶在单分子水平。目前，在单分子的DNA测序领域中有其他小组在工作，但是这些方法是缓慢的，并且DNA作为单链被研究。我们将询问双链DNA，这将使我们能够检测每条链上的甲基化或非甲基化胞嘧啶分子，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，它在特定序列处结合并包裹在双链DNA分子的大沟内。这种人工形式的DNA在结合时被称为三链体，合成的分子也将包含荧光团。当DNA样品用这些形成三链体的寡核苷酸处理后，螺旋将在其沿着的不同点上含有荧光团。我们将DNA样品注入一个小通道，这将导致链的解开和拉直，然后将其穿入光学询问通道。荧光团将被光激发，这在纳米通道内存在纳米结构的情况下将导致荧光强度变化。强度的变化将提供指示不同含胞嘧啶序列的甲基化状态（未甲基化、半甲基化、双甲基化）的代码。检测DNA序列中甲基化和未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理解基因调控。有许多研究领域认为胞嘧啶甲基化在人类中起着重要作用，例如饮食相关疾病，炎症性疾病，胚胎发育等，或在植物中理解环境胁迫的影响。但如上所述，胞嘧啶甲基化对许多生物体都很重要，并且允许分析基因内甲基化模式的技术具有长期商业价值的潜力。首先需要更好地理解DNA甲基化，但这里提出的方法可能会产生诊断工具。

项目成果

Mapping Nanoscale Hotspots with Single-Molecule Emitters Assembled into Plasmonic Nanocavities Using DNA Origami.

• DOI: 10.1021/acs.nanolett.7b04283
• 发表时间: 2018-01-10
• 期刊: Nano letters
• 影响因子: 10.8
• 作者: Chikkaraddy R;Turek VA;Kongsuwan N;Benz F;Carnegie C;van de Goor T;de Nijs B;Demetriadou A;Hess O;Keyser UF;Baumberg JJ
• 通讯作者: Baumberg JJ

Watching individual molecules flex within lipid membranes using SERS.

• DOI: 10.1038/srep05940
• 发表时间: 2014-08-12
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Taylor RW;Benz F;Sigle DO;Bowman RW;Bao P;Roth JS;Heath GR;Evans SD;Baumberg JJ
• 通讯作者: Baumberg JJ