Structural bias in T4 RNA ligase-mediated 3'-adapter ligation.
Structural bias in T4 RNA ligase-mediated 3'-adapter ligation.
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DOI：
10.1093/nar/gkr1263
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发表时间：
2012-04
影响因子：
14.9
通讯作者：
Robb GB
中科院分区：
文献类型：
--
作者：
Zhuang F;Fuchs RT;Sun Z;Zheng Y;Robb GB
T4RNA连接酶通常用于将接头连接到RNA上，但连接效率的巨大差异使检测和定量成为问题。我们开发了一种使用随机RNA和高通量测序相结合的连接选择策略，以深入了解将预腺苷化DNA适配器连接到RNA3‘端的效率差异。分析了T4RNA连接酶在RNA序列、二级结构和RNA-适配器共折叠结构上的偏向，得出结论：T4RNA连接酶在RNA底物上没有表现出显著的一级序列偏好，但对RNA和适配器内的结构特征存在偏见。具体地说，3‘端非结构核苷酸少于3个的RNA以及被预测与接头在不利结构中共折叠的RNA可能连接不良。RNA适配器共折叠结构对连接的影响得到了实验的支持，其中通过设计适配器来改变共折叠结构来改变特定miRNAs的连接效率。此外，我们还发现，使用具有随机化区域的接头可以带来更高的连接效率和更少的连接偏差。我们认为，在包括3‘-接头连接步骤的实验中，使用随机接头可能会改善RNA的表示。
T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3′-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3′-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3′-adapter ligation step.
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Mirbase：MicroRNA基因组学的工具。
影响因子：
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作者：
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