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

本研究针对DNA中的吸氢和电子转移与DNA的局部结构和序列密切相关这一事实，试图通过DNA螺旋沿着的吸氢位点和电子转移效率来探索DNA的局部结构。(1)为了研究尿嘧啶-5-基自由基夺取B型DNA中脱氧核糖C1'和C2' H的性质，我们研究了<Br>脱氧核糖C2'氢被立体专一氘代的六核苷酸d（GCA ^ UGC）_2的光反应。我们发现C2'氧化产物的形成是通过脱氧核糖的C2' α H的限速提取而发生的。(2)研究了5-卤尿嘧啶在不同的生物学重要的DNA局部结构，如A，Z和环中的光反应。我们发现在B DNA中，尿嘧啶-5-基自由基竞争性地夺取脱氧核糖的C1'和C2' α H。在A型DNA中，只得到C1'氧化产物，而在Z型DNA中，观察到前所未有的C2'羟基化。(3)为了研究含~ 3U的DNA的光反应性<Br>，<Br>通过酶促引物延伸法制备了均匀的~ 3U取代的DNA。在<Br>iPrOH存在下，用302 nm的紫外光照射含~（13）U的双链DNA，发现有效的切割发生在<Br>位于5 ′-A ~（13）U ~（13）<Br>U ~<Br>（13）-3 ′位点的~（13）U残基上。

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.