Optimizing splinted ligation of highly structured small RNAs
Optimizing splinted ligation of highly structured small RNAs
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作者：
Kurschat, WC;Müller, J;Helm, M
含有非标准核苷酸的高结构小rna的合成是结构和功能研究的高度兴趣。一般的方法是通过T4 DNA连接酶介导的夹板连接两个或多个RNA片段，每个RNA片段可能包含自己的一组修饰的核苷酸。RNA片段与互补的DNA夹板杂交形成三元连接-胜任复合物（LCC），然后由DNA连接酶翻转。我们研究了LCC及其前体的形成使用尺寸排除色谱结合荧光检测器。通过监测FRET检测lcc中两个花青素染料标记的RNA片段的空间邻近性。观察到的LCC形成和结扎率的相关性建议使用长夹板来稳定LCC。长度增加的夹板寡核苷酸，通常似乎减少了反应混合物中不同杂交中间物种的数量，应用于t4 - dna连接两个高度结构的靶分子，一个是73mer的tRNA，另一个是49mer的合成核酶。一个稳定的LCC可以分离和翻转，结扎效率约为95%。总之，使用长夹板是一种普遍适用的方法，可以克服高度结构化rna的低杂交倾向，从而显著提高连接效率。
The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73mer tRNA, the other a 49mer synthetic ribozyme. A stable LCC could be isolated and turned over with > 95% ligation efficiency. in conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.