Long-read sequencing to detect full-length protein-protein interactions

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Accurate detection of protein-protein interactions is crucial for understanding cellular functions.
• Existing methods for mapping the interactome face limitations in throughput and the ability to capture full-length interactions.

Purpose of the Study:

• To enhance the all-vs.-all sequencing (AVA-Seq) method for determining full-length protein-protein interactions.
• To leverage synthetic DNA technologies and long-read sequencing for improved protein interaction detection.

Main Methods:

• The study adapted the AVA-Seq method using a convergent fusion plasmid design.
• Integration of synthetic DNA technologies and Oxford Nanopore Technologies long-read sequencing (MinION platform).
• Tested 3,115 human protein-protein pairs to identify full-length interactions.

Main Results:

• Successfully recovered 159 protein-protein interactions from 57 full-length human proteins.
• Fifteen of the recovered interactions aligned with known human protein interactions.
• Achieved an 28.6% recovery rate for known interactions from a human gold standard set, consistent with two-hybrid technologies.

Conclusions:

• The enhanced AVA-Seq method provides a low-cost, high-throughput solution for identifying full-length protein-protein interactions.
• This method is accessible to research labs at all stages, facilitating interactome studies.
• Advances in synthetic DNA and long-read sequencing significantly improve protein interaction detection capabilities.