dsDAP: An efficient method for high-abundance DNA-encoded library construction in mammalian cells

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• DNA-encoded libraries are essential for high-throughput screening and functional genomics.
• Constructing high-abundance libraries in mammalian cells presents significant challenges.

Purpose of the Study:

• To introduce dsDNA-assembly-PCR (dsDAP), a novel Gibson-assembly-PCR strategy for efficient DNA-encoded library construction in mammalian cells.
• To investigate the impact of translation initiation sequences (TIS) on protein expression and explore library dilution effects on genotype-phenotype correlations.

Main Methods:

• Developed and applied the dsDNA-assembly-PCR (dsDAP) strategy for DNA-encoded library creation.
• Utilized CRISPR-Cas9 and piggyBac systems for genomic integration in HEK293T cells.
• Analyzed the influence of TIS nucleotides and library dilution on protein expression and genotype-phenotype correlations.

Main Results:

• Demonstrated improved flexibility and efficiency of dsDAP compared to existing methods.
• Confirmed the critical role of specific nucleotides in the TIS, particularly adenine at the -3 position, for high protein expression.
• Showcased the utility of dsDAP for high-abundance library construction in mammalian systems.

Conclusions:

• The dsDAP strategy offers a versatile and efficient solution for constructing high-abundance DNA-encoded libraries in mammalian cells, overcoming limitations of previous techniques.
• This method has significant implications for functional genomics, drug discovery, and gene regulation studies.