Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, Strasbourg, France.
Methods Mol Biol. 2021;2300:203-237. doi: 10.1007/978-1-0716-1386-3_17.
For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (μIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting μIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.
长期以来，人工 RNA 一直是通过体外选择方法来开发的，例如指数富集的配体系统进化（SELEX）。然而，尽管这项技术对于分离特定的高亲和力结合物非常有效，但它不太适合分离优化了更复杂功能的 RNA，例如荧光发射或多次循环催化。由于此类 RNA 理想情况下应该通过筛选方法进行开发，因此常规的微量滴定板测定法很快就变得成本过高。然而，基于液滴的微流控技术的出现最近使我们能够设计基于微流控的体外分隔（μIVC），这是一种强烈微型化和高度并行化的筛选技术，可在一天内对数百万个突变体进行功能筛选，同时使用非常少量的试剂。与高通量测序结合使用时，本章中描述的基于微流控的测序 (μIVC-seq) 管道现在允许以低成本和超高通量模式进行快速半自动筛选。
