Extending the toolbox for RNA biology with SegModTeX: a polymerase-driven method for site-specific and segmental labeling of RNA.

RNA performs a wide range of functions regulated by its structure, dynamics, and often post-transcriptional modifications. While NMR is the leading method for understanding RNA structure and dynamics, it is currently limited by the inability to reduce spectral crowding by efficient segmental labeling. Furthermore, because of the challenging nature of RNA chemistry, the tools being developed to introduce site-specific modifications are increasingly complex and laborious. Here we use a previously designed Tgo DNA polymerase mutant to present SegModTeX - a versatile, one-pot, copy-and-paste approach to address these challenges. By precise, stepwise construction of a diverse set of RNA molecules, we demonstrate the technique to be superior to RNA polymerase driven and ligation methods owing to its substantially high yield, fidelity, and selectivity. We also show the technique to be useful for incorporating some fluorescent- and a wide range of other probes, which significantly extends the toolbox of RNA biology in general.

Extending the toolbox for RNA biology with SegModTeX: A polymerase-driven method for site-specific and segmental labeling of RNA.

RNA performs a wide range of functions regulated by its structure, dynamics, and often post-transcriptional modifications. While NMR is the leading method for understanding RNA structure and dynamics, it is currently limited by the inability to reduce spectral crowding by efficient segmental labeling. Furthermore, because of the challenging nature of RNA chemistry, the tools being developed to introduce site-specific modifications are increasingly complex and laborious. Here we use a previously designed Tgo DNA polymerase mutant to present SegModTeX - a versatile, one-pot, copy-and-paste approach to address these challenges. By precise, stepwise construction of a diverse set of RNA molecules, we demonstrate the technique to be superior to RNA polymerase driven and ligation methods owing to its substantially high yield, fidelity, and selectivity. We also show the technique to be useful for incorporating fluorescent- and a wide range of other probes, which significantly extends the toolbox of RNA biology in general.

Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome.

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome.

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome 1, 2 . The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides 3-6 . To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve 7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.