An extra dimension in nucleic acid sequence recognition

近年来，人类基因组一直是密集研究的对象，所有30亿个信息单位(碱基)的序列现在都是已知的。基因组DNA是由两条互补的链组成的双螺旋结构，由Watson-Crick碱基对(AT和GC)连接在一起，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。传统的DNA序列识别方法是用寡核苷酸(DNA的短单链)探测两条DNA链中的一条，该寡核苷酸只与具有完全互补的碱基序列的相同长度的DNA结合。这在体外是一个有效的过程，因为它很容易通过加热变性DNA双链，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)的非常强大的手段。最近出现了一种实现这一目标的方法(反义技术)。这项技术依赖于一段化学合成的DNA，它与mRNA结合，阻止蛋白质合成。反义可以用来抑制病毒必需蛋白和细胞增殖中的重要蛋白(例如，激酶)的合成。第一批反义药物目前正在进入临床，但进展有限，部分原因是反义治疗的靶点在体内大量产生(对于某些蛋白质，单个细胞中有多达25000个mRNA前体)，如果低于一定水平，存在反馈机制以增加mRNA的产生。因此，完全抑制mRNA几乎是不可能的。然而，在基因水平上的情况是完全不同的；每个基因只有2个拷贝，即使是串联出现的基因，也只有少数几个拷贝存在。通过外部因素直接关闭基因是一个极具吸引力的命题，原则上可以通过阻断双螺旋，使与DNA相互作用的蛋白质不再发挥功能来实现。这将阻止复制和转录，DNA复制的机制和RNA信使(MRNA)控制蛋白质的合成。困难在于开发一种化学试剂，它可以在存在整个人类基因组的情况下与双链DNA的特定区域紧密结合。如果能够开发出这样的试剂，它将在分子生物学、诊断学和医学方面产生深远的影响。反义方法在这里毫无用处，因为它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的第三条链可以插入DNA双链的主槽中，并以序列特异性的方式结合，前提是两条链中的一条富含嘌呤。识别规则很简单；第三链胸腺嘧啶识别A.T，第三链胞嘧啶识别G.C.不幸的是，识别A.T和G.C是不够的，我们还必须有一种识别T.A.和C.G.的方法。此外，三链在生理pH下的稳定性太低，在任何实际应用中都没有价值。天然碱基是不够好的，只有当四个化学修饰的碱基能够被结合到形成三链的寡核苷酸中以使DNA双链能够被序列特异性识别时，三链方法才变得可行。这就是我们研究的目的，我们有四个第一代碱基类似物。下一步是提炼这些分子，以产生一个实用的工作系统，并在诊断和生物医学应用中对它们进行评估。

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.