One of the most effective ways to combat RNA viruses like SARS-CoV-2 is to target the unique structures in their RNA. These viruses rely on highly organized regions in their RNA, called untranslated regions (UTRs), to control essential processes like replication and protein production. Understanding these structures at a detailed level is crucial, and researchers at the University of Chicago have developed a groundbreaking method to do just that: cgSHAPE-seq.

What is cgSHAPE-seq?

cgSHAPE-seq is a sequencing-based technique designed to map the exact binding sites of small molecules on RNA. This innovative method works by introducing a chemical probe that reacts with specific parts of the RNA—specifically the 2’-OH group of ribose. When a small molecule binds to an RNA region, the probe targets that site, creating changes in the RNA sequence. These changes show up as “read-through mutations” during sequencing, revealing the precise location of the binding site.

Development of cgSHAPE-seq and anti-viral RIBOTAC

a RNA secondary structures in SARS-CoV-2 5’ UTR and the pipeline in identification of the ligand binding site and the development of RNA-degrading chimeras. b Principle of cgSHAPE-seq for identifying small molecule binding sites in four steps. Step 1: Synthesis of FAI conjugated chemical probe. Step 2: Chemical-guided acylation at the 2’-OH of ribose at the binding site. Step 3: Reverse transcription in the presence of Mn²⁺ creates single-point mutations at the acylation site. Step 4: Mutational profiling and quantification identify the putative binding sites. c RNA-degrading chimeras (RIBOTACs) recruit RNase L at the target RNA to degrade viral RNAs.

How cgSHAPE-seq Helped Target SARS-CoV-2 RNA

The researchers used cgSHAPE-seq to study the 5’ UTR of the SARS-CoV-2 RNA genome, focusing on a four-way RNA helix called SL5. They discovered that a group of compounds called coumarin derivatives bind to a specific spot within SL5—a bulged guanine (G). This single nucleotide sticks out from the RNA’s structure, making it an ideal target for drug development.

Why cgSHAPE-seq is a Game-Changer

Traditional methods for identifying RNA binding sites often lack precision or are too time-consuming. cgSHAPE-seq offers a direct, accurate way to locate these sites with unparalleled clarity. It allowed the researchers to quickly validate their findings through mutagenesis and lab experiments, saving time and resources.

The insights gained through cgSHAPE-seq enabled scientists to take the next step: designing RNA-degrading molecules that specifically target SL5. These molecules, such as the optimized chimera C64, were shown to reduce viral RNA levels and inhibit live virus replication in lung cells.

Beyond SARS-CoV-2

The applications of cgSHAPE-seq extend far beyond COVID-19. Many RNA viruses have complex UTRs that are critical for their survival. This method could be used to explore RNA structures in other viruses and develop targeted therapies, offering a new approach to combating infectious diseases.

The Future of RNA Research

cgSHAPE-seq represents a leap forward in our ability to study RNA at the molecular level. By pinpointing how small molecules interact with RNA, this method not only aids drug development but also deepens our understanding of RNA biology. In the ongoing fight against viral diseases, tools like cgSHAPE-seq could pave the way for highly targeted and effective treatments.

In a world where RNA viruses pose a constant threat, cgSHAPE-seq offers a powerful new lens to understand—and combat—them.