Enzymatic synthesis of structure-free DNA with pseudo-complementary properties.
Enzymatic synthesis of structure-free DNA with pseudo-complementary properties.
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具有伪平均特性的无结构DNA的酶促合成。
DOI：
10.1093/nar/gkn209
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发表时间：
2008-06
影响因子：
14.9
通讯作者：
Gamper H
中科院分区：
文献类型：
--
作者：
Lahoud G;Timoshchuk V;Lebedev A;de Vega M;Salas M;Arar K;Hou YM;Gamper H
长单链dna和rna在支持与寡核苷酸稳定杂交形成的条件下具有相当大的二级结构。因此，不同的低聚探针可以杂交到相同的目标，效率相差几个数量级。酶促生成任何核酸的无结构单链拷贝而不损害沃森-克里克碱基配对短探针的能力将消除这一问题，并显著提高许多基于寡核苷酸的应用的性能。具有这些特性的合成核酸被定义为伪互补核酸。先前，我们描述了由2-氨基腺嘌呤（nA）和2-硫胸腺嘧啶（sT）碱基组成的伪互补a -t对。虽然nA-T和a - st是稳定的碱基对，但nA-sT对是不匹配的。在这里，我们证明了7-烷基-7-脱氮鸟嘌呤和n4 -烷基胞嘧啶（其中烷基=甲基或乙基）可以与nA和sT结合使用，使DNA在很大程度上无结构和伪互补。这些碱基的脱氧核苷三磷酸（dNTPs）通过选择的嗜中温性和嗜热性DNA聚合酶结合到DNA中，所得到的引物延伸产物与由标准碱基组成的寡核苷酸探针杂交，具有良好的特异性和稳定性。伪互补DNA的合成和性质的进一步优化和表征将导致一个理想的目标，用于长度<25 nt的寡核苷酸探针。
Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson–Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N4-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in length.
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影响因子：
14.9
作者：
Nguyen, HK;Bonfils, E;Thuong, NT
通讯作者：
Thuong, NT
影响因子：
14.9
作者：
Hacia, JG;Woski, SA;Collins, FS
通讯作者：
Collins, FS
影响因子：
14.9
作者：
MIZUSAWA, S;NISHIMURA, S;SEELA, F
通讯作者：
SEELA, F
影响因子：
15
作者：
Kushon, SA;Jordan, JP;Armitage, BA
通讯作者：
Armitage, BA
影响因子：
1.3
作者：
McDougall, MG;Sun, L;Kumar, S
通讯作者：
Kumar, S