Homing endonuclease target determination using SELEX adapted for yeast surface display

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Determining the specific DNA sequence a protein binds to is essential for characterizing DNA-binding proteins and their interactions.
• Predicting target DNA sequences can be challenging or impossible with limited available sequence data, hindering functional analysis of novel proteins like homing endonucleases.

Purpose of the Study:

• To develop a high-throughput, cost-effective method for identifying DNA-binding targets of proteins.
• To enable target site prediction for proteins lacking prior sequence information using a modified SELEX approach.

Main Methods:

• A modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol was developed.
• Yeast surface display technology was integrated with the modified SELEX method.
• The protocol was designed for high-throughput analysis and requires no prior assumptions about the target DNA sequence.

Main Results:

• The modified SELEX method, when combined with yeast surface display, allows for rapid and inexpensive determination of DNA-binding targets.
• This approach is effective for proteins where target site prediction is difficult or not feasible.
• The protocol is easily integrated into existing yeast surface display and SELEX workflows.

Conclusions:

• This modified SELEX technique provides a powerful tool for discovering DNA-binding specificities of proteins.
• It significantly advances the ability to characterize novel DNA-binding proteins and their functions.
• The method offers a valuable, accessible solution for researchers in molecular biology and genetics.