A New Method for Detecting RNA-Protein Binding

Noncoding RNA sequences, including long noncoding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs), and untranslated mRNA regions, fulfill their many diverse functions through direct interactions with RNA-binding proteins (RBPs). RBPs refer to the proteins that bind to RNA, and they have powerful gene regulation capabilities. Except for a few RNAs that can function alone in the form of ribozymes, most RNAs are combined with proteins to form RNA-protein complexes. RBPs play a vital role in regulating life activities such as cell development, differentiation, metabolism, health, and disease and are essential to cellular homeostasis. Therefore, studying the interaction between RNA and protein is the key to exploring the function of RNA. Recent studies have demonstrated that hundreds of novel RBPs lack known RNA-binding domains, showing the complexity and diversity of RNA-protein complexes.

Current methods for studying RNA-protein interactions fall into two categories methods that detect proteins bound to target RNAs (RNA-centric) (shown in Fig.1) and methods that detect RNAs bound to target proteins (protein-centric). RNA-centric assays are generally divided into in vitro and in vivo. In vitro methods are particularly effective for determining which nucleotides and amino acids are involved in known RNA-protein interactions. In vivo approaches are biased towards dynamic studies such as subcellular localization, modifications, or RNA-protein interactions as protein concentrations change. Common protein-centric screens include CLIP-seq, etc., but they have the defect of poor cross-linking efficiency. Although there are many research methods for RBP, researchers are still constantly improving optimization methods to solve more biological problems.

Fig.1 Schematic representation of RNA-centric methods.

A new method for detecting RNA-protein binding that can be used to assess the binding interactions of RNA aptamers with their proteins on a large scale has been developed. This method can transcribe DNA directly on flow cells and measure the binding affinity of RNA aptamers to fluorescently labeled proteins. This method stops the RNA transcription of T7 RNA polymerase upstream of the Tus-bound Ter site by introducing the E. coli replication termination protein (Tus) binding sequence Ter into the system. The transcribed RNA strand remains attached to the RNA polymerase complex due to the abrupt cessation of RNA polymerase activity due to DNA-bound Tus. Then, by introducing fluorescently labeled proteins and binding to the exposed RNA aptamers, a fluorescent signal is generated that can be detected and analyzed.

The Advantages of this platform include:

• Experiments can be completed in a short time, and analysis data can be obtained
• Enough to analyze the interaction between several proteins and millions of different RNAs in a single experiment
• Cell samples can be used directly for detection

Potential Applications

• Detection of RNA-Protein binding
• High-throughput detection possible
• Measure reaction kinetics of RNA-Protein binding

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Reference

1. Ramanathan, M., Porter, D.F. & Khavari, P.A. Methods to study RNA–protein interactions. Nat Methods 16, 225–234 (2019). https://doi.org/10.1038/s41592-019-0330-1