Template-directed photoreversible ligation of deoxyoligonucleotides via 5-vinyldeoxyuridine
Template-directed photoreversible ligation of deoxyoligonucleotides via 5-vinyldeoxyuridine
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DOI：
10.1021/ja993698t
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发表时间：
2000-06-14
影响因子：
15
通讯作者：
Saito, I
中科院分区：
文献类型：
--
作者：
Fujimoto, K;Matsuda, S;Saito, I
虽然已经证明了许多通过天然磷酸二酯键或非天然键进行寡核苷酸模板导向化学连接的方法，但只有少数方法用于光诱导的非酶化学连接。2光化学连接的优点是明显的，避免了需要额外的试剂。通过选择适当的辐照方法，它们的作用在空间和时间上是可控的。因此，光连接方法可用作环状DNA的“双链连接”，2d用作DNA工程和纳米技术的工具，以及用作光调节的诊断和治疗剂。以前报道的DNA光连接方法利用，（i）胸腺嘧啶二聚体的形成，通过在> 290 nm的照射，2a（ii）含有附加的芪类2b或香豆素类2c的DNA的光反应，和（iii）4-硫代胸苷的光加成反应。2d然而，这些方法在实际应用中存在严重的问题，例如光致交联的产率低，2a，d使用对其他组分有害的短波长，2a发生不希望的光交联反应2d和缺乏光可逆性。2b. d因此，似乎非常需要开发在> 360 nm处有效并且优选具有高光可逆性的更有效和清洁的光致电离方法。我们现在希望报道一种高效和可逆的寡脱氧核苷酸（ODNs）的模板导向光连接，它使用5-乙烯基脱氧尿苷，这是远远优于现有的方法上级。我们还证明，几个寡核苷酸同时连接在DNA模板上的366 nm照射，以产生更长的DNA，并得到的连接的DNA定量地恢复到原来的寡核苷酸302 nm照射。用5-乙烯基脱氧尿苷（VU）的β-氰乙基磷酰胺（β-cyanoethylphosphoramide）通过标准自动化DNA合成法制备了5′端含VU的ODN 1。3当ODN 1和ODN 2在没有模板的情况下以366 nm 4照射时，没有观察到光连接产物，但是在模板ODN 3的存在下，产生了预期的连接的12-mer ODN 4，产率为80%，如通过PAGE的光密度测定所确定的（图1，泳道4）。5增加照射时间12 h，光连接ODN的产率可提高至96%。在模板ODN 3的存在下，ODN 1和ODN 2的光照射混合物的HPLC分析表明连接的ODN 4的干净和有效的形成，伴随着ODN 1和2的消失。ESI-TOF MS表明ODN 4是ODN 1与ODN 2的连接产物。7酶消化分离的ODN 4，然后进行HPLC分析，表明形成了2：5：3比例的dC、dG和dT以及新产物。ESI-TOF MS等光谱数据表明，该新产物为dU-VU加合物5，它是在碱性磷酸酶作用下由最初形成的dC-VU加合物脱氨基而得。8事实上，光连接的ODN 4在pH 5.0-9.0甚至在90 ℃下都是稳定的，但是通过加入碱性磷酸酶使ODN 4快速脱氨基以产生含dU-VU的ODN。这些结果清楚地表明，dU-VU加合物5是在酶消化过程中由最初形成的dC-VU加合物脱氨基产生的。通过HPLC纯化获得的5的结构基于包括1H-1H COSY和NOESY的光谱数据被指定为顺式-顺式[2+ 2]加合物5。8对ODN 1、ODN 2和模板ODN 3组成的双链体的分子模拟研究表明，ODN 1的5′-末端VU的乙烯基在其s-反式构象中与ODN 3的5，6-双键堆叠。
While many methods for template-directed chemical ligation of oligonucleotides via a native phosphodiester bond or non-native linkages have been demonstrated, 1 there are only a few methods for photoinduced non-enzymatic chemical ligation. 2 The merit of the photochemical ligation avoiding the need for additional reagents is obvious. Their actions are controllable within space and time by the choice of proper irradiation methods. Thus, the photoligation methods can be used as “photopadlocking” of circular DNAs, 2d as a tool for DNA engineering and nanotechnology, and as photoregulated diagnostic and therapeutic agents. Previously reported methods for DNA photoligation utilized,(i) the thymine dimer formation by irradiation at> 290 nm, 2a (ii) photoreactions of DNA containing appended stilbenes2b or coumarins2c, and (iii) the photoaddition reaction of 4-thiothymidine. 2d However, these methods have serious problems for practical applications, such as low yields of photoligation, 2a, d the use of short wavelength that is injurious to other components, 2a the occurrence of undesirable photo-cross-linking reaction2d and the lack of photoreversibility. 2b. d It seems therefore highly desirable to develop more efficient and clean photoligation methods that are effective at> 360 nm and preferably possess high photoreversibility. We now wish to report a highly efficient and reversible template-directed photoligation of oligodeoxynucleotides (ODNs) which uses 5-vinyldeoxyuridine and which is far superior to the existing methods. We also demonstrate that several oligonucleotides are simultaneously ligated on a DNA template by 366 nm irradiation to produce a longer DNA, and the resulting ligated DNA is quantitatively reverted to the original oligonucleotides by 302 nm irradiation. ODN 1 containing 5-vinyldeoxyuridine (VU) at the 5′ end was prepared by standard automated DNA synthesis using β-cyanoethylphosphoramide of VU. 3 When ODN 1 and ODN 2 were irradiated at 366 nm4 in the absence of template, no photoligation product was observed, but in the presence of template ODN 3, the expected ligated 12-mer ODN 4 was produced in 80% yield as determined by densitometric assay of PAGE (Figure 1, lane 4). 5 The yield of the photoligated ODN increased up to 96% by increasing irradiation time for 12 h. HPLC analysis of the photoirradiated mixture of ODN 1 and ODN 2 in the presence of template ODN 3 indicated a clean and efficient formation of ligated ODN 4 with concomitant disappearance of ODNs 1 and 2. ESI-TOF MS indicated that ODN 4 is a ligated product of ODN 1 with ODN 2. 7 Enzymatic digestion of isolated ODN 4 followed by HPLC analysis indicated the formation of dC, dG, and dT in a ratio of 2: 5: 3 together with a new product. Spectroscopic data including ESI-TOF MS indicated that this new product was dU-VU adduct 5 which was derived from deamination of initially formed dC-VU adduct by the action of alkaline phosphatase. 8 In fact, photoligated ODN 4 was stable at pH 5.0-9.0 even at 90 C, but ODN 4 was rapidly deaminated by adding alkaline phophatase to produce dU-VU containing ODN. These results clearly indicated that dU-VU adduct 5 was produced from deaminaion of initially formed dC-VU adduct during enzymatic digestion. The structure of 5 obtained by HPLC purification was assigned as a cis-syn [2+ 2] adduct 5 on the basis of spectroscopic data including 1H-1H COSY and NOESY. 8 Molecular modeling studies of the duplex consisting of ODN 1, ODN 2, and template ODN 3 suggested that the vinyl group of the 5′-terminal VU of ODN 1 in its s-trans conformation is stacked with the 5, 6-double bond of the 3 …