Cyclodextrin-peptide conjugates for sequence specific DNA binding.

Area of Science:

• Synthetic biology
• Molecular biology
• Chemical synthesis

Background:

• Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences.
• Developing synthetic TF models with precise DNA recognition is crucial for understanding gene regulation and for biotechnological applications.
• Existing synthetic TF models often face challenges in synthesis complexity and DNA binding efficiency.

Purpose of the Study:

• To design and synthesize novel synthetic models of bZIP transcription factors capable of specific DNA recognition.
• To develop an efficient and convergent synthetic strategy for constructing these TF models.
• To evaluate the DNA binding affinity and specificity of the synthesized constructs.

Main Methods:

• Copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) for conjugating peptides to cyclodextrins.
• Synthesis of azide-functionalized α, β, and γ-cyclodextrin derivatives.
• Conjugation of basic region Leucine Zipper (bZIP) peptides to cyclodextrin scaffolds.
• DNA binding assays to determine sequence specificity and affinity (nM range).

Main Results:

• Successfully synthesized bZIP TF models by conjugating cationic peptides to azide-functionalized cyclodextrins.
• Achieved efficient and simultaneous conjugation of two long, unprotected peptides to cyclodextrin-bis azide derivatives.
• Demonstrated specific recognition of cognate DNA sequences by the synthetic TF models with nanomolar affinities.
• The developed synthetic route is convergent and easier compared to previous TF models.

Conclusions:

• The novel synthetic bZIP TF models exhibit high specificity and affinity for their target DNA sequences.
• The employed CuAAC-mediated conjugation provides a versatile and efficient method for constructing complex synthetic TFs.
• These findings offer a promising platform for developing advanced gene regulation tools and synthetic biology applications.