Advantages of 2′-O-methyl oligoribonucleotide probes for detecting RNA targets
Advantages of 2′-O-methyl oligoribonucleotide probes for detecting RNA targets
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DOI：
10.1093/nar/26.9.2224
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发表时间：
1998-05-01
影响因子：
14.9
通讯作者：
Becker, MM
中科院分区：
文献类型：
--
作者：
Majlessi, M;Nelson, NC;Becker, MM
我们比较了含有2 '-O-甲基核苷酸或2'-脱氧核苷酸的寡核糖核苷酸探针在检测核酸靶标的测定中的各种动力学和熔解性质。在所有测试长度（8-26个碱基）下，2 '-O-甲基寡核糖核苷酸探针比相应的2'-脱氧寡核糖核苷酸探针更快地结合到RNA靶标，并且具有高得多的解链温度（T-m值）。两种探针的T-m值随着长度增加至约19个碱基，在16个碱基或更少的长度处观察到2 ′-O-甲基寡核糖核苷酸探针和2 ′-脱氧寡核糖核苷酸探针之间的T-m的最大差异。与RNA靶相比，2 ′-O-甲基寡核糖核苷酸探针与DNA靶的结合更慢，并且与相应的2 ′-脱氧寡核糖核苷酸探针具有相同的T-m。由于它们在与RNA结合时大大增强的T-m，2 '-O-甲基寡核糖核苷酸探针可以有效地与结构化RNA分子的双链区域结合。17个碱基的2 ′-O-甲基寡核糖核苷酸探针能够结合rRNA的双链区域，而相同的17个碱基的2 ′-脱氧寡核糖核苷酸探针不能。由于它们在与RNA靶结合时的T-m增强，较短的2 ′-O-甲基寡核糖核苷酸探针可以在测定中代替较长的2 ′-脱氧寡核糖核苷酸探针使用，导致匹配和错配RNA靶之间的区分增强，12个碱基的2 '-O-甲基寡核糖核苷酸探针具有与19个碱基的2'-O-甲基寡核糖核苷酸探针相同的T-m。当与匹配的RNA靶结合时，2 ′-脱氧寡核糖核苷酸探针的T-m降低，但当与含有1或2个错配碱基的RNA靶结合时，2 ′-脱氧寡核糖核苷酸探针的T-m降低比2 ′-脱氧寡核糖核苷酸探针的T-m降低大得多。T-m增加，杂交动力学更快，结合结构化靶的能力和2 ′-O-甲基寡核糖核苷酸探针的增加的特异性使它们在检测RNA靶的测定中的使用优于相应的2 ′-脱氧寡核糖核苷酸上级。
We have compared various kinetic and melting properties of oligoribonucleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotides with regard to their use in assays for the detection of nucleic acid targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (T-m values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). T-m values of both probes increased with length up to -19 bases, with maximal differences in T-m between 2'-O-methyl and 2'-deoxy oligoribonucleotide probes observed at lengths of 16 bases or less. In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes bound more slowly and with the same T-m to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced T-m when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a double-stranded region of rRNA whereas the same 17 base 2'-deoxy oligoribonucleotide probe did not. Due to their enhanced T-m when bound to RNA targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resulting in enhanced discrimination between matched and mismatched RNA targets, A 12 base 2'-O-methyl oligoribonucleotide probe had the same T-m as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in T-m than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases, The increased T-m, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.