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Xeno nucleic acid probes mediated methylation-specific PCR for single-base resolution analysis of N6-methyladenosine in RNAs
Analyst ( IF 3.6 ) Pub Date : 2021-08-20 , DOI: 10.1039/d1an01291f
Qinli Pu 1, 2 , Hongyan Yu 1 , Xi Zhou 1 , Junjie Li 1 , Yujun Yang 1 , Ting Wang 1 , Fugang Li 3 , Shangchun Sheng 4 , Guoming Xie 1
Affiliation  

Reliable and cost-effective quantification of RNA modifications at a specific gene locus is essential to elucidate the pathogenic mechanism encoded by RNA epigenetics. Current methods to quantify N6-methyladenosine (m6A) at specific sites can hardly satisfy the requirement of clinical application because epigenetic information is easily lost through polymerase chain reaction (PCR) assay or other isothermal amplification methods unless tedious pretreatment is applied. Herein, we propose a simple xeno nucleic acid (XNA) as a blocker probe to mediate the methylation specific reverse transcription quantitative polymerase chain reaction (MsRT-qPCR) assay to directly magnify the minor differences between epigenetic bases and unmodified bases in RNA. Strand displacement reactions selectively initiated between the reverse transcription primer (RT-primer) and the XNA probe at the m6A template given the affinity differences between the blocker probes and the m6A-modified RNA (m6A-RNA) and unmodified RNA (A-RNA). Thus, preferential amplification of m6A-RNA was allowed. Integration of a well-established oligo-modified Fe3O4@UiO-66-NH4 allowed purification of mRNA and lncRNA from cellular total RNA samples and greatly reduced the non-specific interference of m6A detection in real samples. Multiple specific sites of m6A in mRNA and lncRNA samples are also successfully quantified. The XNA probe-based m6A assay required only common and available lab equipment and materials, which can be applied in m6A-related fundamental studies and clinical diagnosis.

中文翻译:

异种核酸探针介导的甲基化特异性 PCR 用于 RNA 中 N6-甲基腺苷的单碱基分辨率分析

对特定基因位点的 RNA 修饰进行可靠且经济高效的量化对于阐明 RNA 表观遗传学编码的致病机制至关重要。当前量化 N 6 的方法特定位点的-甲基腺苷(m6A)很难满足临床应用的要求,因为除非进行繁琐的预处理,否则通过聚合酶链反应(PCR)检测或其他等温扩增方法很容易丢失表观遗传信息。在此,我们提出了一种简单的异种核酸 (XNA) 作为阻断探针,以介导甲基化特异性逆转录定量聚合酶链反应 (MsRT-qPCR) 测定,以直接放大 RNA 中表观遗传碱基和未修饰碱基之间的微小差异。鉴于阻断探针与 m6A 修饰的 RNA (m6A-RNA) 和未修饰的 RNA (A-RNA) 之间的亲和力差异,在逆转录引物 (RT-primer) 和 XNA 探针之间在 m6A 模板上选择性启动链置换反应. 因此,允许优先扩增 m6A-RNA。整合成熟的低聚修饰 Fe3 O 4 @UiO-66-NH 4允许从细胞总RNA样品中纯化mRNA和lncRNA,并大大降低了m6A检测在实际样品中的非特异性干扰。mRNA 和 lncRNA 样品中 m6A 的多个特定位点也被成功量化。基于 XNA 探针的 m6A 检测只需要常见和可用的实验室设备和材料,可应用于 m6A 相关的基础研究和临床诊断。
更新日期:2021-09-22
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