A new tool greatly improves scientists' ability to identify and study proteins that regulate gene activity in cells, according to research led by Weill Cornell Medicine investigators. The technology should enable and enhance investigations in both fundamental biology and disease research.
The activity of a gene is often regulated—switched on, sped up, slowed down, switched off—by one or more proteins that bind to DNA to exert their effect. However, identifying these DNA-binding proteins has been challenging due to the lack of a precise method. In their study, reported Sept. 29 in the Proceedings of the National Academy of Sciences, the researchers developed a molecular tool that can be targeted to virtually any spot on the genome to capture any protein that lies nearby, allowing the protein's identification. The team demonstrated the power of their new tool by using it to discover new protein regulators of human stem cell-related genes.
"We expect this to be useful as a very general laboratory tool, and we already plan to use it for research on specific disorders, including type 1 diabetes," said study co-senior author Dr. Shuibing Chen, the Kilts Family Professor of Surgery, director of the Center for Genomic Health and a member of the Hartman Institute for Therapeutic Organ Regeneration at Weill Cornell Medicine.
The study's other co-senior author was Dr. Peter Schultz, the Skaggs Presidential Chair of Chemistry and CEO and President at Scripps Research.
The new tool, which the researchers have dubbed SCOPE, has two key elements. One is a protein incorporating a "guide RNA" that can be designed to bind to virtually any site on the genome. The other key element is a special amino acid—a building block of proteins—that when hit with a flash of ultraviolet light will form a strong and enduring bond to any protein in close proximity. Researchers using the tool can relatively easily isolate the resulting SCOPE-bound protein and identify it using a standard set of methods called mass spectrometry.
The special amino acid used in SCOPE has the further remarkable property that it is based on an amino acid found only in some ancient, single-celled microorganisms called archaea. Because of this evolutionary distance, it has essentially zero natural reactivity with other amino acids in mammalian and even bacterial cells—it becomes reactive only when exposed to UV light.
"This reduces the chances of unwanted interactions between the SCOPE tool and other proteins, which effectively gives SCOPE a high sensitivity, enabling it to detect proteins that are DNA-bound only weakly and/or transiently," said study first author Dr. Jiajun Zhu, a postdoctoral researcher in the Chen laboratory.
The use of the special amino acid, known as AbK, was pioneered by the Schultz laboratory, where Dr. Chen did her graduate research, in work aimed at developing other laboratory tools.
The SCOPE tool is meant to be engineered into the DNA of any cell type, including stem cells; it thus assembles and operates internally in the cell to bind proteins at any genomic site specified by the guide RNA.
In their study, the team used SCOPE to illuminate the DNA-binding roles of three proteins in human embryonic stem cells. They showed that two of the proteins work to maintain these stem cells in their immature, stem-like state, while the third helps induce the differentiation of the stem cells into more mature cell types.
Drs. Chen and Zhu and their colleagues now hope to use SCOPE to uncover gene-regulating proteins in other cell types and in specific disease contexts, including cardiomyocytes in heart arrhythmias, insulin-producing pancreatic cells in type 1 diabetes, and neurons in neurodegenerative disorders.
Dr. Shuibing Chen is the founder of Oncobeat, Inc. and iOrganBio, Inc.
This work was supported by the National Institute of Diabetes, Digestive and Kidney Diseases, part of the National Institutes of Health, through grant numbers R01DK137517, R01DK124463 and R01DK130454. Additional support was provided by the Department of Surgery at Weill Cornell Medicine.
