DNA duplex stabilization in crowded polyanion solutions

The melting temperature of duplex DNA is much higher in polyanions than in non-ionic polymers with similar ionic strength, suggesting an additional electrostatic contribution on top of the excluded volume effect.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

DNA-Functionalized Gold Nanoparticles in Macromolecularly Crowded Polymer Solutions

This document is the Accepted Manuscript version of a Published Work that appeared in final form in The Journal of Physical Chemistry B, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jp310662mDNA-functionalized gold nanoparticles (AuNPs) are one of the most commonly used reagents in nanobiotechnology. They are important not only for practical applications in analytical chemistry and drug delivery, but also for fundamental understanding of nanoscience. For biological samples such as blood serum or for intracellular applications, the effects of crowded cellular proteins and nucleic acids need to be considered. The thermodynamic effect of crowding is to induce nanoparticle aggregation. But before such aggregation can take place, there might also be a depletion repulsive barrier. Polyethylene glycol (PEG) is one of the most frequently used polymers to mimic the crowded cellular environment. We show herein that while DNA-functionalized AuNPs are very stable in buffer (e.g., no PEG) and citrate-capped AuNPs are very stable in PEG, DNA-functionalized AuNPs are unstable in PEG and are easily aggregated. Although such aggregation in PEG is mediated by DNA, no sharp melting transition typical for DNA-linked AuNPs is observed. We attribute this broad melting to depletion force instead of DNA base pairing. The effects of PEG molecular weight, concentration and temperature have been studied in detail and we also find an interesting PEG phase separation and AuNP partition into the water-rich phase at high temperature.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles

mRNA lipid nanoparticles (LNPs) have emerged as powerful modalities for gene therapies to control cancer and infectious and immune diseases. Despite the escalating interest in mRNA-LNPs over the past few decades, endosomal entrapment of delivered mRNAs vastly impedes therapeutic developments. In addition, the molecular mechanism of LNP-mediated mRNA delivery is poorly understood to guide further improvement through rational design. To tackle these challenges, we characterized LNP-mediated mRNA delivery using a library of small molecules targeting endosomal trafficking. We found that the expression of delivered mRNAs is greatly enhanced via inhibition of endocytic recycling in cells and in live mice. One of the most potent small molecules, endosidine 5 (ES5), interferes with recycling endosomes through Annexin A6, thereby promoting the release and expression of mRNA into the cytoplasm. Together, these findings suggest that targeting endosomal trafficking with small molecules is a viable strategy to potentiate the efficacy of mRNA-LNPs