Probing DNA local structure by photoirradiation and application toward gene therapy.

在本研究中，我们着眼于DNA中的氢转移和电子转移在很大程度上依赖于DNA的局部结构和序列，并试图通过DNA螺旋上的氢转移位置和电子转移效率来探索DNA局部结构的方法。(1)为了研究B型DNA中脱氧核糖C_1‘和尿酰-5-基C_2’H的性质，我们研究了六核苷酸d(GCA^<br>UGC)_2的光反应，其中一个脱氧核糖结构的C2氢是立体特异性的。我们发现C2‘氧化产物的形成是通过对脱氧核糖C2’αH的限速提取进行的。(2)研究了A、Z和loop等多种生物重要DNA局部结构中包含的5-氟尿嘧啶的光反应。我们发现在B DNA中，尿酰-5-基竞争性地提取脱氧核糖的C_1‘和C_2’αH。在A型DNA中只有C_1‘氧化产物，而在Z型DNA中观察到了前所未有的C2’羟基化。(3)为了考察含U的DNA的光反应活性，用酶引物法制备了均一的U取代DNA。在iPrOH存在下，在302 nm处照射含U的双链DNA，发现有效的切割发生在位于5‘-A^<br>U^<br>U-3’位的^<br>U残基。

In this research we focused on the fact that the hydrogen abstraction and electron transfer in DNA significantly depend on the local structures and sequence of DNA and we tried to find out a method to probe DNA local structure by the site of hydrogen abstraction and efficiency of electron transfer along DNA helix.(1) In order to investigate the nature of deoxyribose C1' and C2' H abstraction by uracilyl-5-yl radical in B form DNA,we have examined the photoreaction of hexanucleotides d (GCA ^<Br>UGC) _2 in which one of the C2' hydrogens of the deoxyribose moiety was stereospecifically deuterated. We found that the formation of C2' oxidation product occurs via a rate-limiting abstraction of C2'alpha H of the deoxyribose.(2) Photoreaction of 5-halouracil contained in a various biologically important DNA local structures, such as A,Z,and loop was examined. We found that in B DNA uracilyl-5-yl radical competitively abstract C1' and C2'alpha H of deoxyribose. In A form DNA,only C1' oxidation product was obtained whereas in Z-form DNA unprecedented C2' hydroxylation was oberved.(3) In order to investigate the photoreactivity of ^<Br>U-containing DNA,uniformly ^<Br>U-substituted DNA was prepared by enzymatic primer extension method. The ^<Br>U-containing duplex DNA was irradiated at 302nm in the presence of iPrOH.It was found that the efficient cleavages occur at ^<Br>U residues located in 5'-A ^<Br>U^<Br>U-3' sites.