An extra dimension in nucleic acid sequence recognition

核酸序列识别的额外维度

• 批准号: BB/D003318/1
• 负责人: Tom Brown
• 金额: $ 42.64万
• 依托单位: University of Southampton
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD003318%2F1
• 关键词: extra; dimension; nucleic; acid; sequence

In recent years the human genome has been the subject of intense study and the sequence of all 3 billion units of information (bases) is now known. Genomic DNA is a double helix consisting of two complementary strands held together by Watson-Crick base pairs (AT and GC), and the ability to recognise specific DNA sequences forms the basis of molecular biology, molecular genetics and diagnostics. The conventional approach to DNA sequence recognition is to probe one of the two DNA strands with an oligonucleotide (short single strand of DNA) which will only bind to an equivalent length of DNA with an exactly complementary base sequence. This is an efficient process in vitro as it is easy to denature the DNA duplex by heat, thereby allowing an oligonucleotide probe to bind to one of the strands. In theory the ability to interfere with the biological function of DNA would be a very powerful means of killing viruses and curing certain diseases such as cancer. A method of achieving this objective (antisense technology) has recently emerged. This technology relies upon a chemically synthesised piece of DNA binding to mRNA and preventing protein synthesis. Antisense can be used to inhibit the synthesis of essential viral proteins and important proteins in cell proliferation (e.g. kinases). The first antisense drugs are now coming into the clinic but progress has been limited, partly because the target for antisense therapy (mRNA) is produced in large quantities in vivo (for some proteins there are as many as 25,000 mRNA precursors in a single cell) and feedback mechanisms exits to increase mRNA production if it falls below a certain level. Therefore it is almost impossible to completely inhibit mRNA. The situation at the level of the gene, however, is totally different; there are only 2 copies of each gene and even in the case of genes that occur in tandem, only a handful of copies exist. Directly switching off genes by an external agent is an extremely attractive proposition and in principle it could be achieved by blocking the double helix so that the proteins that interact with DNA can no longer function. This would prevent replication and transcription, the mechanisms by which DNA is copied and the RNA messengers (mRNA) control the synthesis of proteins. The difficulty lies in developing a chemical agent that can bind tightly to a specific region of duplex DNA in the presence of the entire human genome. If such an agent could be developed it would have profound implications in molecular biology, diagnostics and medicine. The antisense approach is of no use here as it is not possible to separate the two strands of genomic DNA in vivo to allow the antisense oligonucleotide to bind. However, Nature offers us clues to solving the problem. It has been known for some time that a third strand of natural DNA can fit into the major groove of the DNA duplex and bind in a sequence-specific manner, provided that one of the two strands of the duplex is purine-rich. The recognition rules are simple; a third strand thymine recognizes A.T and a third strand cytosine recognises G.C. Unfortunately, recognition of A.T and G.C is not sufficient, we must also have a means of recognising T.A. and C.G. In addition, the stability of triplexes at physiological pH is too low to be of value in any practical applications. Natural bases are simply not good enough and the triplex approach will only become feasible if four chemically modified bases can be developed for incorporation into triplex forming oligonucleotides to enable the sequence specific recognition of DNA duplexes. This is the aim of our research and we have four first generation base analogues. The next stage is to refine these molecules to produce a practical working system and evaluate them in diagnostic and biomedical applications.

近年来，人类基因组一直是密集研究的主题，所有30亿个信息单位（碱基）的序列现在已经知道了。基因组DNA是由沃森-克里克碱基对（AT和GC）连接在一起的两条互补链组成的双螺旋，识别特定DNA序列的能力构成了分子生物学，分子遗传学和诊断学的基础。DNA序列识别的常规方法是用寡核苷酸（DNA的短单链）探测两条DNA链中的一条，该寡核苷酸仅结合具有完全互补的碱基序列的相等长度的DNA。这是一个有效的体外过程，因为它很容易通过加热使DNA双链体变性，从而允许寡核苷酸探针结合到其中一条链上。从理论上讲，干扰DNA生物功能的能力将是杀死病毒和治愈某些疾病（如癌症）的一种非常强大的手段。最近出现了实现这一目标的方法（反义技术）。该技术依赖于化学合成的DNA片段与mRNA结合并阻止蛋白质合成。反义可用于抑制必需病毒蛋白和细胞增殖中的重要蛋白（例如激酶）的合成。第一个反义药物现在进入临床，但进展有限，部分原因是反义治疗的靶点（mRNA）在体内大量产生（对于某些蛋白质，单个细胞中有多达25，000个mRNA前体），如果mRNA福尔斯低于一定水平，则存在反馈机制以增加mRNA产量。因此，几乎不可能完全抑制mRNA。然而，在基因水平上的情况完全不同;每个基因只有2个拷贝，即使在串联发生的基因的情况下，也只有少数拷贝存在。通过外部试剂直接关闭基因是一个非常有吸引力的提议，原则上可以通过阻断双螺旋来实现，这样与DNA相互作用的蛋白质就不再起作用。这将阻止复制和转录，DNA复制和RNA信使（mRNA）控制蛋白质合成的机制。困难在于开发一种化学试剂，可以在整个人类基因组存在的情况下与双链DNA的特定区域紧密结合。如果能够开发出这样一种药物，它将在分子生物学、诊断学和医学方面产生深远的影响。反义方法在此没有用，因为不可能在体内分离基因组DNA的两条链以允许反义寡核苷酸结合。然而，大自然为我们提供了解决问题的线索。一段时间以来，人们已经知道，天然DNA的第三条链可以适合DNA双链体的大沟，并以序列特异性方式结合，前提是双链体的两条链之一富含嘌呤。识别规则很简单：第三条胸腺嘧啶识别A.T，第三条胞嘧啶识别G. C。不幸的是，仅仅承认A.T和G. C是不够的，我们还必须有一种承认T. A的方法。和重心此外，三链体在生理pH下的稳定性太低，在任何实际应用中都没有价值。天然碱基根本不够好，并且三链体方法只有在可以开发四种化学修饰的碱基用于掺入三链体形成寡核苷酸中以使得能够序列特异性识别DNA双链体时才变得可行。这是我们研究的目的，我们有四个第一代碱基类似物。下一阶段是改进这些分子以产生实际工作系统，并在诊断和生物医学应用中对其进行评估。

项目成果

DNA triple-helix formation at target sites containing duplex mismatches.

在含有双链体错配的靶位点形成 DNA 三螺旋。

• DOI: 10.1016/j.bpc.2006.04.016
• 发表时间: 2006
• 期刊: Biophysical chemistry
• 影响因子: 3.8
• 作者: Rusling DA

Photoinduced crosslinking of double-helical DNA by psoralen covalently linked to a triple helix-forming oligonucleotide under near-physiological conditions.

在接近生理条件下，补骨脂素与形成三螺旋的寡核苷酸共价连接，导致双螺旋 DNA 发生光诱导交联。

• DOI: 10.1080/15257770701508554
• 发表时间: 2007
• 期刊: Nucleosides, nucleotides & nucleic acids
• 作者: Li H

DNA triplex formation with 5-dimethylaminopropargyl deoxyuridine.

• DOI: 10.1093/nar/gkn1060
• 发表时间: 2009-03
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Rusling DA;Peng G;Srinivasan N;Fox KR;Brown T
• 通讯作者: Brown T

Synthesis of anthraquinone oligonucleotides for triplex stabilization.

用于三链体稳定的蒽醌寡核苷酸的合成。

• DOI: 10.1080/15257770701506491
• 发表时间: 2007
• 期刊: Nucleosides, nucleotides & nucleic acids
• 作者: Zhao Z