Researchers can now more accurately and precisely target specific proteins in yeast, mammalian cells and mice to study how knocking down specific protein traits can influence physical manifestation in a cell or organism. The researchers published their results in Nature Communications.
"Conditional gene knockout and small interfering RNA, which is used to silence proteins without knocking them out completely, has been employed in many studies," said Masato T. Kanemaki, lead researcher from the National Institute of Genetics in the Research Organization of Information and Systems. "However, these technologies are not ideal for studying highly dynamic processes, such as cell cycle, differentiation, or neural activity, because of the slow rate of depletion of the protein of interest."
Kanemaki and his team had previously developed an approach called the AID system, which uses a small protein tag, known as a degron, fused to proteins to induce degradation. "The original AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin," Kanemaki said. "These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and apply this method to mice."
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"In this paper, we describe the AID2 system, which overcomes all the drawbacks of the original AID system," Kanemaki said, noting that they did not detect leaky degradation with the system, the degradation was quicker, and the required dose of auxin was much lower.
To establish the AID2 system, the researchers employed what is known as a bump-and-hole strategy to create an empty space in a mutant version of a plant protein called TIR1, that recognizes and induces the degradation of degron-fused proteins. An auxin analog can bind directly to the TIR1 mutant and initiate the degradation process. The researchers found that depletion could be induced at a concentration about 670 times lower than in the original system.