VRK1 co-delivery mitigates DNA clustering by BAF in TFAMoplex transfection.

Area of Science:

• Biotechnology
• Gene Delivery
• Molecular Biology

Background:

• Non-viral gene delivery faces challenges with DNA aggregation by barrier-to-autointegration factor (BAF) in the cytoplasm.
• This BAF-mediated clustering hinders DNA translocation through nuclear pores, limiting transfection efficiency in interphase cells.
• Previous attempts to fuse vaccinia-related kinase 1 (VRK1) to TFAM (TFAMoplex) to inhibit BAF did not resolve DNA clustering.

Purpose of the Study:

• To engineer a modified TFAMoplex system (SpyTFAMoplex) using a SpyTag/SpyCatcher linker to effectively inhibit BAF clustering.
• To investigate the impact of active versus inactive VRK1 on BAF clustering and subsequent gene delivery efficiency.
• To explore the potential of co-delivering enzymes with DNA for improved gene delivery strategies.

Main Methods:

• Development of SpyTFAMoplex, linking VRK1 to TFAM via SpyTag/SpyCatcher.
• Quantitative image analysis of BAF clustering in enhanced green fluorescent protein (EGFP)-BAF overexpressing HeLa cells.
• Comparison of transfection efficiency between SpyTFAMoplex (active VRK1) and dSpyTFAMoplex (inactive VRK1 mutant).

Main Results:

• SpyTFAMoplex successfully counteracted cytoplasmic BAF clustering compared to controls.
• A version with inactive VRK1 (dSpyTFAMoplex) showed twice the number of BAF clusters per cell.
• Active VRK1 in SpyTFAMoplex led to a significant decrease (-72%) in reporter gene expression (mean fluorescence intensity) in HeLa cells.

Conclusions:

• The SpyTFAMoplex system demonstrates the ability to inhibit BAF-mediated DNA clustering.
• Active VRK1, while reducing BAF clusters, negatively impacts exogenous DNA transfection efficiency.
• VRK1 may interfere with the DNA-binding ability of BAF, potentially affecting the mitotic nuclear entry pathway of DNA, necessitating further vector engineering.