Gamper H B, Parekh H, Rice M C, Bruner M, Youkey H, Kmiec E B
Department of Biological Sciences, University of Delaware, 105 Wolf Hall, Newark, DE 19716, USA.
Nucleic Acids Res. 2000 Nov 1;28(21):4332-9. doi: 10.1093/nar/28.21.4332.
Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.
嵌合寡核苷酸（嵌合体）由RNA和DNA碱基组成，通过互补折叠成双发夹构象，已被证明可改变或修复植物和动物基因组中的单个碱基。已知嵌合体内一段不间断的DNA碱基在序列改变中起作用，而RNA残基有助于复合物的稳定性。在本研究中，将两条链分开，希望确定每条链在转化中所起的作用。使用一系列由所有RNA或DNA残基以及各种混合物组成的单链寡核苷酸，几种新结构已成为核苷酸转化中的可行分子。当使用哺乳动物和植物细胞提取物以及细菌中的遗传读出测定法时，发现含有一定数量硫代磷酸酯主链修饰的单链寡核苷酸在基因修复过程中比原始嵌合体结构更具活性。发现含有完全修饰主链的单链寡核苷酸具有低修复活性，实际上会诱导突变。还发现含有不同长度修饰RNA碱基（2'-O-甲基）的分子活性较低。综上所述，这些结果证实了嵌合体DNA链进行核苷酸转化的方向性，并进一步提出了一种新型的、修饰的单链DNA分子，其可在植物和动物无细胞提取物中指导转化。
