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Preparation of Novel Modified Oligonucleotides by Chemic-Enzymatic Method and Their Application for the production of New DNA Catalyst

化学酶法制备新型修饰寡核苷酸及其在新型DNA催化剂生产中的应用

基本信息

批准号：
13480183
负责人：
SAWAI Hiroaki
金额：
$ 9.28万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

We prepared various novel C5-substituted 2'-deoxyuridine-5'-triphosphates, which have an amino group, a carboxyl group, imidazole derivatives, pyridine, biotin, chelating groups, fluorescence and amino acids at a C5-terminal via a linker. Many of them could work as a substrate for several thermophilic DNA polymerases. B-Family DNA polymerases obtained from thermophilic archaeas, such as KOD dash, Vent(exo-) and Pwo DNA polymerase, could accept the C5-substituted deoxyuridine derivatives and gave the corresponding modified DNA by PCR. On the other hand, A-family DNA polymerases obtained from thermophilic bacteria, such as Taq DNA polymerase, could not accept these modified 2'-deoxyuridine derivatives at all. The C5 substituted deoxyuridine derivatives bearing an anionic carboxyl group, a bulky group with a short linker or sulfur-containing compounds were a poor substrate for any DNA polymerases. Post-synthetic functionalization of the modified DNA obtained by PCR extends the varieties of the resulting modified DNA. We also prepared 2'-deoxycytidine derivatives bearing several functional groups at C5-position and assessed their substrate properties for the thermophilic DNA polymerases. KOD dash and Vent(exo-) DNA polymerases could accept some C5-substituted 2'-deoxycytidines forming the cytidine-modified DNA by PCR. Double-modified DNA containing the modified 2'-deoxyuridine and modified 2'-deoxycytidine derivatives could be prepared enzymatically by using KOD dash or Vent(exo-) DNA polymerase. We applied the modified DNA synthesis by PCR for the production of a new modified DNA aptamer and a modified DNA catalyst by combination with in vitro selection method. The modified DNA aptamers, which bind with sialyllactose, thalidomide or aspartame were produced, and their binding abilities and sequences were examined to asses the roles of the functional groups in the modified DNA for the binding.

我们制备了各种新型C5取代的2'-脱氧尿苷-5'-三磷酸盐，其通过连接体在C5末端具有氨基、羧基、咪唑衍生物、吡啶、生物素、螯合基团、荧光和氨基酸。其中许多可以作为几种嗜热 DNA 聚合酶的底物。从嗜热古菌中获得的B家族DNA聚合酶，如KOD dash、Vent(exo-)和Pwo DNA聚合酶，可以接受C5取代的脱氧尿苷衍生物，并通过PCR得到相应的修饰DNA。另一方面，从嗜热细菌获得的A家族DNA聚合酶，例如Taq DNA聚合酶，根本不能接受这些修饰的2'-脱氧尿苷衍生物。带有阴离子羧基、具有短接头的大基团或含硫化合物的 C5 取代脱氧尿苷衍生物对于任何 DNA 聚合酶来说都是不良底物。通过 PCR 获得的修饰 DNA 的合成后功能化扩展了所得修饰 DNA 的品种。我们还制备了在 C5 位带有多个官能团的 2'-脱氧胞苷衍生物，并评估了它们对于嗜热 DNA 聚合酶的底物特性。 KOD dash 和 Vent(exo-) DNA 聚合酶可以接受一些 C5 取代的 2'-脱氧胞苷，通过 PCR 形成胞苷修饰的 DNA。含有修饰的2'-脱氧尿苷和修饰的2'-脱氧胞苷衍生物的双修饰DNA可以通过使用KOD dash或Vent(exo-) DNA聚合酶酶促制备。我们应用PCR合成修饰DNA并结合体外选择方法生产新的修饰DNA适体和修饰DNA催化剂。制备了与唾液酸乳糖、沙利度胺或阿斯巴甜结合的修饰 DNA 适体，并检查了它们的结合能力和序列，以评估修饰 DNA 中官能团对结合的作用。