Kelly Skyler L, Strobel Eric J
Department of Biological Sciences, The University at Buffalo, Buffalo, NY, 14260, USA.
Nat Commun. 2025 Mar 10;16(1):2350. doi: 10.1038/s41467-025-57415-3.
RNA can fold into structures that mediate diverse cellular functions. Understanding how RNA primary sequence directs the formation of functional structures requires methods that can comprehensively assess how changes in an RNA sequence affect its structure and function. Here we have developed a platform for performing high-throughput cotranscriptional RNA biochemical assays, called Transcription Elongation Complex display (TECdisplay). TECdisplay measures RNA function by fractionating a TEC library based on the activity of cotranscriptionally displayed nascent RNA. In this way, RNA function is measured as the distribution of template DNA molecules between fractions of the transcription reaction. This approach circumvents typical RNA sequencing library preparation steps that can cause technical bias. We used TECdisplay to characterize the transcription antitermination activity of >1 million variants of the Clostridium beijerinckii pfl ZTP riboswitch designed to perturb steps within its cotranscriptional folding pathway. Our findings establish TECdisplay as an accessible platform for high-throughput RNA biochemical assays.
RNA能够折叠成介导多种细胞功能的结构。要理解RNA一级序列如何指导功能结构的形成，就需要能够全面评估RNA序列变化如何影响其结构和功能的方法。在此，我们开发了一个用于进行高通量共转录RNA生化分析的平台，称为转录延伸复合物展示（TECdisplay）。TECdisplay通过基于共转录展示的新生RNA的活性对TEC文库进行分级分离来测量RNA功能。通过这种方式，RNA功能被测量为转录反应各组分之间模板DNA分子的分布。这种方法规避了可能导致技术偏差的典型RNA测序文库制备步骤。我们使用TECdisplay对拜氏梭菌pfl ZTP核糖开关的100多万个变体的转录抗终止活性进行了表征，这些变体旨在干扰其共转录折叠途径中的步骤。我们的研究结果确立了TECdisplay作为高通量RNA生化分析的一个易用平台。
