Allerson C R, Verdine G L
Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
Chem Biol. 1995 Oct;2(10):667-75. doi: 10.1016/1074-5521(95)90030-6.
Several factors impede the elucidation of RNA structure and function by X-ray and NMR methods, including the complexity of folded RNA motifs, the tendency of RNA to aggregate, and its ability to fold into multiple isomeric structures. The ability to constrain the process of RNA folding to give a single, homogeneous product would assist these investigations. We therefore set out to develop a synthetic procedure for the site-specific insertion of a disulfide crosslink into oligoribonucleotides. We also examined the ability of a crosslinked species to serve as a substrate for ricin, an RNA glycosylase.
A convertible nucleoside derivative (C) suitable for the site-specific introduction of N4-alkylcytidine residues into RNA has been developed. The corresponding C phosphoramidite was employed in the synthesis of an 8-mer oligonucleotide, 5'-CGGA-GACG-3', which was then efficiently converted to an 8-mer containing two S-protected N4-(2-thioethyl)C residues. Upon deprotection and air oxidation, the 8-mer efficiently formed an intramolecular disulfide bond, yielding a GAGA tetraloop presented on a two-base-pair CpG disulfide crosslinked ministem. We show that this ministem-loop is an excellent substrate for ricin. Control 8-mers lacking the disulfide crosslink were substantially poorer substrates for ricin.
The nucleoside chemistry described here should be generally useful for the site-specific introduction of a range of non-native functional groups into RNA. We have used this chemistry to constrain an RNA ministem through introduction of an intrahelical disulfide crosslink. That this tetraloop substrate linked to a two base-pair ministem is efficiently processed by ricin is clear evidence that ricin makes all of its energetically favorable contacts to the extreme end of the stem-loop structure, and that the two base pairs of the stem abutting the loop remain intact during recognition and processing by ricin.
多种因素阻碍了通过X射线和核磁共振方法阐明RNA的结构和功能，这些因素包括折叠RNA基序的复杂性、RNA聚集的倾向以及其折叠成多种异构体结构的能力。限制RNA折叠过程以产生单一、均匀产物的能力将有助于这些研究。因此，我们着手开发一种用于将二硫键交联位点特异性插入寡核糖核苷酸的合成方法。我们还研究了交联物种作为RNA糖基化酶蓖麻毒素底物的能力。
已开发出一种适用于将N4 - 烷基胞苷残基位点特异性引入RNA的可转换核苷衍生物（C）。相应的C亚磷酰胺用于合成8聚体寡核苷酸5'-CGGA - GACG - 3'，然后将其有效地转化为含有两个S保护的N4 - （2 - 硫代乙基）C残基的8聚体。脱保护并经空气氧化后，该8聚体有效地形成了分子内二硫键，产生了一个由两碱基对CpG二硫键交联的小茎呈现的GAGA四环。我们表明，这个小茎环是蓖麻毒素的优良底物。缺乏二硫键交联的对照8聚体作为蓖麻毒素的底物则差得多。
本文所述的核苷化学方法通常应可用于将一系列非天然官能团位点特异性引入RNA。我们已利用这种化学方法通过引入螺旋内二硫键交联来限制RNA小茎。与两碱基对小茎相连的这个四环底物能被蓖麻毒素有效加工，这清楚地证明蓖麻毒素与茎环结构的最末端形成了所有能量上有利的接触，并且在蓖麻毒素识别和加工过程中，与环相邻的茎的两个碱基对保持完整。
