Constructing Nucleic Acid Delivering Lipoproteoplexes from Coiled-Coil Supercharged Protein and Cationic Liposomes

Thomas, Joseph; Sun, Jonathan; Montclare, Jin Kim

doi:10.1007/978-1-0716-3469-1_14

Cited by 1 publication

(2 citation statements)

References 29 publications

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“…Expression and purification of the mutant CSP variants were conducted with minimal adjustments to our previously described protocol 20 . Plasmids were transformed via heat shock into chemically competent AF‐IQ E. coli cells and incubated on tryptic soy agar plates containing 0.2‐μ g/μL ampicillin and 0.034 μg/μL chloramphenicol to grow for 12–16 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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Supercharged coiled‐coil protein with N‐terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex

Sun,

Thomas,

Monkovic

et al. 2024

Journal of Peptide Science

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Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non‐palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non‐viral materials, while offering safer and non‐cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled‐coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C‐terminal decahistidine tags on α‐helical secondary structure. Cross‐correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

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Section: Methodsmentioning

confidence: 99%

“…Transfection complexes were prepared in a previously described two‐step process 16,20 . The desired CSP variant was mixed with siGFP (220 ng) at a 3:1 protein/siGFP w/w ratio in pure deionized water for 30 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%