Gamper Howard B, Arar Khalil, Gewirtz Alan, Hou Ya-Ming
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Biochemistry. 2006 Jun 6;45(22):6978-86. doi: 10.1021/bi0600392.
The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays.
长单链DNA和RNA中二级结构的存在对短寡核苷酸探针（<20聚体）的实际应用构成了严重障碍。在此，我们表明，在一组独特的dNTP类似物存在的情况下，高度结构化DNA的复制会导致合成二级结构水平显著降低的子代DNA。这种由2-氨基腺嘌呤、2-硫代胸腺嘧啶、7-脱氮鸟嘌呤和胞嘧啶碱基组成的复制DNA，很容易被平铺的8聚体LNA和15聚体DNA探针识别，而相同DNA的未修饰版本则无法被识别。重要的是，虽然碱基类似物增强了探针与靶标的稳定性，但它们并没有显著降低碱基配对的特异性。无结构DNA靶标的可用性应有助于短寡核苷酸探针的使用，并促进通用寡核苷酸微阵列的发展。
