SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts. SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts

We developed a Copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC) to capture NAD-RNAs without RNA degradation. We examined the specificity of CuAAC and SPAAC reactions towards NAD+ vs. m7G, and found that both prefer NAD+ but also act on m7G. We show that m7G-capped RNA can be immuno-depleted, allowing for the specific identification of NAD-RNA via the SPAAC reaction and sequencing, which we name SPAAC-NAD-seq. Subjecting Arabidopsis RNA to both the original NAD captureSeq and SPAAC-NAD-seq, we found that more NAD+-capped RNA was identified by the latter, particularly those with low abundance. This led to the discovery of new gene ontology terms such as starch biosynthsis, intracellular protein transport and response to cadmium stress associated with genes that produce NAD-RNA. Furthermore, reads were uniformly distributed along gene bodies, which suggested that SPAAC-NAD-seq retained full-length sequence information. SPAAC-NAD-seq enables specific and efficient discovery of NAD-RNA in prokaryotes, and when combined with m7G-RNA depletion, in eukaryotes. Overall design: NAD capped transcripts profiled by CuAAC-NAD-seq and SPAAC-NAD-seq in Arabidopsis

本研究开发了一种无铜张力促进叠氮-炔环加成反应（SPAAC），用于在不发生RNA降解的前提下捕获NAD修饰RNA（NAD-RNA）。我们评估了铜催化叠氮-炔环加成反应（CuAAC）与SPAAC对烟酰胺腺嘌呤二核苷酸（NAD+）与7-甲基鸟苷（m7G）的反应特异性，结果显示二者均偏好作用于NAD+，但同样可与m7G发生反应。研究证实，7-甲基鸟苷（m7G）帽化RNA可通过免疫清除法去除，从而可借助SPAAC反应与测序技术实现NAD-RNA的特异性鉴定，我们将该方法命名为SPAAC-NAD-seq。将拟南芥RNA分别通过原始的NAD捕获测序（NAD captureSeq）与SPAAC-NAD-seq进行处理后，我们发现后者可鉴定出更多的NAD+帽化RNA，尤其是其中的低丰度物种。这一结果助力我们挖掘出与NAD-RNA编码基因相关的全新基因本体（GO）术语，包括淀粉生物合成、细胞内蛋白质运输以及镉胁迫响应等。此外，测序读段在基因体区域呈均匀分布，这表明SPAAC-NAD-seq可保留完整的序列信息。SPAAC-NAD-seq可在原核生物中实现NAD-RNA的特异性高效鉴定，而与m7G RNA清除技术联用时，也可应用于真核生物的NAD-RNA研究。实验整体设计：采用CuAAC-NAD-seq与SPAAC-NAD-seq对拟南芥中的NAD帽化转录本进行谱分析。