Sequestered RNA Rolling Circle Amplification within MS2 Virus-Like Particles for Drug Delivery
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Braunstein, Melissa
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Abstract
Virus-like particles (VLPs) are ideal vessels for targeted drug delivery due to their biocompatibility, structural uniformity, and ability to package therapeutic cargo. Traditional encapsulation methods, such as co-expression of VLPs with target nucleic acids or disassembly/reassembly in the presence of cargo, often suffer from contamination and limited loading efficiency. To overcome these challenges, we present a reaction scheme that encapsulates polymerases and ssDNA templates within MS2 VLPs with added (d)NTPs to perform rolling circle amplification (RCA) in situ. Our lab has previously been successful experimenting with this idea using phi29 DNA polymerase. In this study, we explore the use of T7 RNA polymerase for RCA with an ssDNA template encoding the T7 forward promoter and the Baby Spinach RNA aptamer. We also attempted to engineer MS2 VLP pores via FG-loop mutations to prevent nucleic acid efflux, hypothesizing that steric and electrostatic modifications would enhance RNA retention. Results showed limited assembly of mutants using IPTG induction, and further optimization of expression and purification systems must be investigated to produce enough VLPs for experimentation. Furthermore, improved template design is necessary for more consistent RNA production through RCA. This work demonstrates the potential for more efficient cargo-loading strategies that could lead to new RNA therapeutic strategies.
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2025-05-22
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