Interaction proteomics analysis to provide insight into TFAMoplex-mediated transfection

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Delivery Systems

Background:

• TFAMoplex, a DNA nanoparticle transfection agent, previously utilized mitochondrial transcription factor A (TFAM) protein, bacterial phospholipase, and vaccinia-related kinase 1 (VRK1).
• VRK1 enhanced transfection efficiency via an unknown mechanism.
• The study explored protein engineering to optimize TFAMoplex for gene delivery.

Purpose of the Study:

• To replace VRK1 in TFAMoplex with dynein light chain RP3 for enhanced cytosolic transport via dynein motor complex tethering.
• To investigate the interaction between TFAM-RP3 fusion protein and dynein intermediate chains.
• To compare cytosolic protein interactions of different TFAMoplex variants using a proteomics-based assay.

Main Methods:

• Fusion protein construction (TFAM-RP3).
• Binding kinetics analysis of TFAM-RP3 with dynein intermediate chains.
• Proteomics-based assay for comparing cytosolic protein interactions of TFAMoplex variants.
• Incorporation of leucine-rich repeat-containing protein 59 (LRRC59) into TFAMoplex.

Main Results:

• TFAM-RP3 fusion protein confirmed interaction with dynein components.
• VRK1-containing TFAMoplex showed significant shifts in protein interactors, particularly nucleolar proteins.
• LRRC59-modified TFAMoplex demonstrated improved transfection properties compared to RP3-modified and original VRK1-containing systems.

Conclusions:

• Protein engineering of TFAMoplex by incorporating LRRC59 enhances gene delivery efficiency.
• The study provides insights into optimizing TFAMoplex for effective gene delivery systems.
• Understanding protein interactions is key to designing advanced transfection agents.