Three RNA Microenvironments Detected in Fluxional Gene Delivery Polyplex Nanoassemblies

Area of Science:

• Biochemistry
• Polymer Science
• Molecular Biology

Background:

• Polyplexes are complexes of polymers and nucleic acids, crucial for gene delivery.
• Understanding the microenvironment within polyplexes is key to optimizing their function.
• Fluorescein (FL) is a sensitive fluorescent probe for microenvironmental changes.

Purpose of the Study:

• To investigate the local microenvironments of RNA within polycationic polymer (POCP)/RNA polyplexes.
• To utilize the prototropic and solvatochromatic properties of fluorescein (FL) for microenvironment detection.
• To correlate polyplex microenvironments with polymer structure and charge ratios.

Main Methods:

• Conjugation of fluorescein to HIV-1 transactivation response element (TAR) RNA (TAR-FL).
• Formation of polyplexes using various POCPs and TAR-FL.
• Analysis of fluorescence properties of TAR-FL to probe local pH and solvent characteristics.
• Varying the polymer nitrogen to RNA phosphate (N:P) ratio to assess microenvironment changes.

Main Results:

• Identified distinct microenvironments for polyplex-bound RNA, including pH-dependent and acidic regions.
• Observed RNA experiencing local pH changes influenced by the POCP nitrogen to RNA phosphate (N:P) ratio.
• Detected fluorophore/fluorophore quenching indicating close RNA packing.
• Demonstrated that the magnitude of microenvironmental changes is polymer-dependent.

Conclusions:

• Polyplex microenvironments are complex and influenced by polymer type and N:P ratio.
• Local pH and solvent properties of bound RNA can be characterized using FL.
• This approach elucidates the functional hierarchy of polyplex-bound oligonucleotides and characterizes POCPs.