Covalent crosslinking systems are applied to CLIP technology to improve the purity of the resulting protein-RNA complex.

131,《》(Nucleic Acids Research):SpyCLIP:an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy。
the innovative application of SpyTag-SpyCatcher, a covalent crosslinking system, into CLIP technology, greatly reduces the difficulty of CLIP technology and improves the specificity of the data obtained.
study also redesigned the CLIP library process to accommodate the requirements of automated operations and high-throughput applications.
more than a thousand rna binding proteins are known to exist, and many proteins previously thought to be non-bound RNA have also recently been found to have RNA binding capabilities, and the boundRNA plays an important role in regulating the activity of these protein machines.
CLIP is one of the most advanced methods for studying protein-RNA interactions in the body.
the method has been improved and produced a variety of variants, but the existing CLIP version sits on highly specific antibodies, the experimental steps are complicated, usually using radioisotope marking, the data obtained is poorly repeatable, can not be applied on a large scale.
therefore, the development of new methods that are simple, efficient and suitable for high-throughput applications is a key and imperative for a systematic understanding of protein-RNA interactions.
the study used a new purification strategy based on SpyTag-SpyCatcher, a co-price cross-linked system, which greatly improved the purity of the resulting protein-RNA complex.
innovative design of the PROCESS of CLIP library at the same time, avoiding the efficiency-limiting steps of the traditional CLIP method to limit large-scale operation and extensive loss of starting materials. in addition,
, a common input library was introduced for background deduction, further enhancing the specificity of the CLIP method.
these core improvements not only avoid the reliance on antibodies from existing CLIP methods, improve the signal-to-noise ratio and repeatability of the data, and significantly reduce the difficulty and duration of operations, enabling the SpyCLIP method to adapt to the needs of high-throughput research. Using spyCLIP, a new tool
, researchers studied the binding maps of the containing sub-binding proteins PTBP1 and RBFOX2 in mammalian cells and found that SpyCLIP can capture their shear regulatory sites in a real and efficient way; SpyCLIP was found to be able to capture its interaction with histone mRNA with very high specificity, and new non-histone binding objects were found, and the binding map of the microRNA effector protein AGO2 was studied to compare and reveal the mechanisms and characteristics of 3'non-translated and coding regions microRNA target sites.
SpyDescribeshows outstanding sensitivity and specificity in a wide range of RNA binding proteins, providing a new, simple and easy-to-use, high-precision tool for large-scale, systematic study of protein-RNA interactions.
Wu Ligang is the paper's communication author, associate researcher Zhao Ya and doctoral student Zhang Wei are co-first authors of the paper.
the research work is supported by funds from the Chinese Academy of Sciences, the Fund Committee and the Ministry of Science and Technology.
Source: Institute of Biochemistry and Cell Biology.