Kore Anilkumar R, Hodeib Melissa, Hu Zhongting
An Applied Biosystems Business, Bioorganic Chemistry Division, Ambion, Inc., Austin, Texas, USA.
Nucleosides Nucleotides Nucleic Acids. 2008 Jan;27(1):1-17. doi: 10.1080/15257770701571693.
Locked nucleic acids (LNA) are being applied in hybridization studies, but current locked nucleotides cannot be transcribed into RNA probes. Here, the authors report the use of a new synthetic locked nucleotide, locMeCytidine-5'-triphosphate (LNA-mCTP), for hybridization study. This synthetic LNA-mCTP can be transcribed into a short ( approximately 30-nt) RNA probe. Dot blot hybridization on nylon membrane suggested that the short (33)P-LNA RNA probes had strong binding affinity to target oligonucleotides and its detection sensitivity was approximately approximately 1000 miRNAs in a 20- to 30-mum (diameter) dot area. On tissue sections, the differential expression pattern of mir-124 within different tissue regions revealed by short (33)P- LNA RNA probes correlated well to that analyzed by real-time RT-PCR. In addition, the specific cellular distribution of vasoactive intestinal polypeptide mRNAs in the mouse brain was the same using a 30-nt (33)P-LNA RNA probe and a 1.5-kb (33)P-RNA probe. These results suggested the high hybridization specificity of the small LNA-RNA probes to target small RNAs. Finally, the authors applied (33)P-LNA probes to detect miRNA let-7C expression in human cancer tissues. Let-7C was clearly present in lung, prostate, and colon cancers but undetectable in ovary and thyroid cancer samples. These results suggested that this miRNA detection method provides an alternative tool to study the cellular distribution of miRNAs in tissues.
锁核酸（LNA）正应用于杂交研究，但目前的锁核苷酸无法转录成RNA探针。在此，作者报道了一种新的合成锁核苷酸——锁甲基胞苷-5'-三磷酸（LNA-mCTP）在杂交研究中的应用。这种合成的LNA-mCTP可以转录成短的（约30个核苷酸）RNA探针。尼龙膜上的点杂交表明，短的（33）P-LNA RNA探针与靶寡核苷酸具有很强的结合亲和力，其检测灵敏度在直径为20至30微米的点区域内约为1000个微小RNA。在组织切片上，短的（33）P-LNA RNA探针揭示的不同组织区域内mir-124的差异表达模式与实时RT-PCR分析的结果相关性良好。此外，使用30个核苷酸的（33）P-LNA RNA探针和1.5千碱基的（33）P-RNA探针，小鼠脑中血管活性肠多肽mRNA的特定细胞分布是相同的。这些结果表明小LNA-RNA探针与靶小RNA具有高杂交特异性。最后，作者应用（33）P-LNA探针检测人类癌症组织中微小RNA let-7C的表达。let-7C在肺癌、前列腺癌和结肠癌中明显存在，但在卵巢癌和甲状腺癌样本中无法检测到。这些结果表明，这种微小RNA检测方法为研究组织中微小RNA的细胞分布提供了一种替代工具。
