Department of Microbial Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom.
Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom.
Sci Rep. 2017 Jul 26;7(1):6542. doi: 10.1038/s41598-017-06970-x.
Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of "smart materials", namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation.
虽然对单细胞的转录组和蛋白质组进行分析已经可行，但对翻译组的分析，即涉及与核糖体结合进行蛋白质合成的所有信使 RNA（mRNA），仍然是一个需要数百万个细胞的复杂过程。在这里，我们报告了“智能材料”的产生和使用，即分子印迹聚合物（MIPs），以方便从仅 1000 个细胞中分离核糖体和翻译的 mRNA。特别是，我们表明，基于水凝胶的核糖体印迹聚合物可以从人、猿和鼠的细胞提取物中回收核糖体和相关的 mRNA，但不会选择性地富集酵母核糖体，从而表现出选择性。此外，核糖体印迹聚合物能够灵敏地测量需要比当前方法少 1000 倍细胞的 mRNA 翻译调控事件。这些结果首次证明了 MIPs 适合选择性地回收核糖体等核糖核蛋白复合物，为灵敏检测基因调控提供了一种新方法。
