Synthesis and physical and physiological properties of 4′-thioRNA:: application to post-modification of RNA aptamer toward NF-κB
Synthesis and physical and physiological properties of 4′-thioRNA:: application to post-modification of RNA aptamer toward NF-κB
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DOI：
10.1093/nar/gkh705
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Matsuda, A
中科院分区：
文献类型：
--
作者：
Hoshika, S;Minakawa, N;Matsuda, A
我们在此报告了基于我们的合成方案，通过Pummerer反应制备4'-硫脲，-胞苷，-腺苷和-鸟苷磷酰胺的全部细节。按照标准RNA合成方法制备了含有4种4′-硫核糖核苷单元的完全修饰的4′-硫核糖核酸。采用紫外熔化和差示扫描量热法（DSC）测定了一系列双相化合物的T、、值和热力学参数。得到的双链物的总体热稳定性顺序为4'-thioRNA:4'-thioRNA >> 4'-thioRNA:RNA > RNA:RNA > RNA:DNA > 4'-thioRNA:DNA。此外，研究还表明，由4′-硫核糖核酸组成的双链化合物的稳定性的主要因素是焓性质。4'-thioRNA:RNA和4'-thioRNA:4'-thioRNA的CD谱线与a -构象双链的CD谱线相似，4'-thioRNA:DNA的CD谱线也与a -构象双链相似。4′-硫核糖核酸在人血清中的稳定性是天然RNA的600倍。在相同的条件下，RNA:RNA和4'-thioRNA: 4'-thioRNA双链都没有被消化。第一个例子后修饰RNA适体的4'-硫核糖核苷单位被证明。对适体thioRNA3的完全修饰导致其结合活性完全丧失。相比之下，在结合位点以外的位置进行修饰是可以容忍的，而不会失去结合活性。修饰后的RNA适体thioRNA5具有热稳定性和抗核酸酶酶切性。希望本文的研究结果能够为4'-thioRNA作为新一代人工RNA的发展做出贡献。
We report herein full details of the preparation of 4'-thiouridine, -cytidine, -adenosine and -guanosine phosphoramidites based on our synthetic protocol via the Pummerer reaction. Fully modified 4'-thioRNAs containing four kinds of 4'-thioribonucleoside units were prepared according to the standard RNA synthesis. The T,, values and thermodynamic parameters of a series of duplexes were determined by UV melting and differential scanning calorimetry (DSC) measurements. The resulting overall order of thermal stabilities for the duplexes was 4'-thioRNA:4'-thioRNA >> 4'-thioRNA:RNA > RNA:RNA > RNA:DNA > 4'-thioRNA:DNA. In addition, it was shown that the dominant factor in the stability of the duplexes consisting of 4'-thioRNA was enthalpic in character. The CD spectra of not only 4'-thioRNA:RNA and 4'-thioRNA:4'-thioRNA but also 4'-thioRNA:DNA were all similar to those of duplexes in the A-conformation. The stability of 4'-thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA:RNA nor the 4'-thioRNA: 4'-thioRNA duplexes were digested under the same conditions. The first example of a post-modification of an RNA aptamer by 4'-thioribonucleoside units was demonstrated. Full modification of the aptamer thioRNA3 resulted in complete loss of binding activity. In contrast, modifications at positions other than the binding site were tolerated without loss of binding activity. The post-modified RNA aptamer thioRNA5 was thermally stabilized and resistant toward nuclease digestion. The results presented in this paper will, it is hoped, contribute to the development of 4'-thioRNA as a new generation of artificial RNA.