用于检测RNA靶标的2'-O-甲基寡核糖核苷酸探针的优势。
Advantages of 2'-O-methyl oligoribonucleotide probes for detecting RNA targets.
作者信息
Majlessi M, Nelson N C, Becker M M
机构信息
Gen-Probe Inc., 10210 Genetic Center Drive, San Diego, CA 91212-4362, USA.
出版信息
Nucleic Acids Res. 1998 May 1;26(9):2224-9. doi: 10.1093/nar/26.9.2224.
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 (Tm values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). Tm values of both probes increased with length up to approximately 19 bases, with maximal differences in Tm 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 Tm to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced Tm 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 Tm 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 Tm as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in Tm than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases. The increased Tm, 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.
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用于检测核酸靶标的分析中的各种动力学和熔解特性。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与RNA靶标的结合速度更快，熔解温度（Tm值）比相应的2'-脱氧寡核糖核苷酸探针高得多。两种探针的Tm值均随长度增加至约19个碱基而升高，在16个碱基或更短的长度下观察到2'-O-甲基和2'-脱氧寡核糖核苷酸探针之间的Tm最大差异。与RNA靶标相反，2'-O-甲基寡核糖核苷酸探针与DNA靶标的结合更慢，且与相应的2'-脱氧寡核糖核苷酸探针具有相同的Tm。由于其与RNA结合时Tm大大提高，2'-O-甲基寡核糖核苷酸探针能够有效结合到结构化RNA分子的双链区域。一个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则不能。由于其与RNA靶标结合时Tm提高，较短的2'-O-甲基寡核糖核苷酸探针可用于分析中替代较长的2'-脱氧寡核糖核苷酸探针，从而增强对匹配和错配RNA靶标的区分能力。当与匹配的RNA靶标结合时，一个12个碱基的2'-O-甲基寡核糖核苷酸探针与一个19个碱基的2'-脱氧寡核糖核苷酸探针具有相同的Tm，但当与含有1个或2个错配碱基的RNA靶标结合时，其Tm的降低幅度比2'-脱氧寡核糖核苷酸探针大得多。2'-O-甲基寡核糖核苷酸探针Tm的提高、杂交动力学加快、结合结构化靶标的能力以及特异性增强，使其在检测RNA靶标的分析中优于相应的2'-脱氧寡核糖核苷酸。
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