Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase
Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase
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DOI：
10.1017/s1355838201001698
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发表时间：
2001-02-01
期刊：
影响因子：
4.5
通讯作者：
Lyamichev, VI
中科院分区：
文献类型：
--
作者：
Allawi, HT;Dong, F;Lyamichev, VI
描述了一种快速简单的方法，用于确定RNA中的可接近位点，该方法不依赖于靶RNA的长度，并且不需要RNA标记。在该方法中，允许靶RNA与DNA寡核苷酸的序列随机化文库杂交，所述DNA寡核苷酸在其5 '端连接至共同标签序列。用逆转录酶延伸退火的寡核苷酸，然后通过使用PCR扩增延伸的产物，所述PCR使用对应于标签序列的引物和对靶RNA序列特异的第二引物。我们使用RT-PCR产物的长度和RNA特异性引物的结合位点的位置的组合来确定RNA分子的哪些区域是RNA可延伸位点，即可用于寡核苷酸结合和延伸的位点。然后，我们采用这种逆转录与随机寡核苷酸文库（RT-ROL）方法来确定四种mRNA靶点上的可接近位点，所述四种mRNA靶点是人活化的res（ha-ras）、人细胞间粘附分子-1（ICAM-1）、兔β-珠蛋白和人干扰素-γ（IFN-γ）。我们的研究结果与其他研究人员使用RNase H切割或与寡核苷酸阵列杂交来确定这些靶点上的可访问位点的结果一致。此外，我们发现良好的相关性网站时，我们比较了可延伸的网站的位置确定的RT-ROL与杂交位点的有效反义寡核苷酸的ICAM-1 mRNA的反义抑制研究。最后，我们讨论了RNA可延伸位点和RNA可及性之间的关系。
A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated res (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta -globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility.