Centre for Integrative Biology, University of Trento, Via Sommarive, 9 Povo, Italy; IMMAGINA Biotechnology s.r.l., Via alla cascata 56/c, Povo, Italy.
Centre for Integrative Biology, University of Trento, Via Sommarive, 9 Povo, Italy.
Cell Rep. 2018 Oct 23;25(4):1097-1108.e5. doi: 10.1016/j.celrep.2018.09.084.
Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.
核糖体图谱分析（ribosome profiling，或 Ribo-seq）是基于对核糖体保护免受核酸酶消化的 RNA 片段进行大规模测序。由于该方法具有独特的能力，可以提供核糖体沿转录本移动的位置信息，因此可用于阐明翻译的机制方面。然而，目前的核糖体图谱分析方法缺乏区分活性翻译核糖体或无活性核糖体保护的片段的能力。为了克服这一可能的限制，我们开发了 RiboLace，这是一种基于原始含有嘌呤霉素的分子的方法，该分子能够通过无抗体和无标签的下拉方法分离活性核糖体。RiboLace 速度快，在低输入量的情况下可靠工作，并且易于在体外和体内快速应用，从而以单核苷酸分辨率生成活性核糖体足迹的全局快照。
