An extra dimension in nucleic acid sequence recognition

近年来，人类基因组一直是研究的热点，目前已知所有30亿个信息单位（碱基）的序列。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结构，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（短单链DNA）探测两条DNA链中的一条，该DNA链只与具有完全互补碱基序列的等效长度的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使DNA双链变性，从而允许寡核苷酸探针结合到其中一条链上。理论上，干扰DNA生物功能的能力将是杀死病毒和治疗某些疾病（如癌症）的一种非常有力的手段。最近出现了一种实现这一目标的方法（反义技术）。这项技术依赖于化学合成的DNA片段与mRNA结合并阻止蛋白质合成。反义蛋白可用于抑制病毒必需蛋白和细胞增殖中重要蛋白（如激酶）的合成。第一批反义药物现在正在进入临床，但进展有限，部分原因是反义治疗的靶标（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的产量低于一定水平，则存在反馈机制来增加mRNA的产量。因此完全抑制mRNA几乎是不可能的。然而，基因层面的情况则完全不同；每个基因只有2个拷贝，即使在基因串联出现的情况下，也只有少数拷贝存在。通过外部因素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结构来实现，这样与DNA相互作用的蛋白质就不能再起作用了。这将阻止复制和转录，即DNA复制和RNA信使（mRNA）控制蛋白质合成的机制。难点在于开发一种化学制剂，能够在整个人类基因组存在的情况下与双工DNA的特定区域紧密结合。如果这种药剂能够被开发出来，将对分子生物学、诊断学和医学产生深远的影响。反义方法在这里没有用处，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这个问题的线索。一段时间以来，人们已经知道，天然DNA的第三条链可以进入DNA双链的主要凹槽，并以特定序列的方式结合，前提是双链的两条链中有一条富含嘌呤。识别规则简单；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，三联体在生理pH值下的稳定性太低，在任何实际应用中都没有价值。天然碱基根本不够好，只有开发出四种化学修饰的碱基，将其整合到三联体形成的寡核苷酸中，才能实现对DNA双链的序列特异性识别，三联体方法才可行。这是我们研究的目的，我们有四个第一代碱基类似物。下一阶段是对这些分子进行细化，以产生一个实际的工作系统，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.