Linear PEI is a cationic polymer commonly used for complexing DNA into nanoparticles for cell-transfection and gene-therapy applications. The polymer has closely-spaced amines with weak-base protonation capacity, and a hydrophobic backbone that is kept unaggregated by intra-chain repulsion. As a result, in solution PEI exhibits multiple buffering mechanisms, and polyelectrolyte states that shift between aggregated and free forms. We studied the interplay between the aggregation and protonation behavior of 2.5 kDa linear PEI by pH probing, vapor pressure osmometry, dynamic light scattering, and ninhydrin assay. Our results indicate that:At neutral pH, the PEI chains are associated and the addition of NaCl initially reduces and then increases the extent of association.The aggregate form is uncollapsed and co-exists with the free chains.PEI buffering occurs due to continuous or discontinuous charging between stalled states.Ninhydrin assay tracks the number of unprotonated amines in PEI.The size of PEI-DNA complexes is not significantly affected by the free vs. aggregated state of the PEI polymer.Despite its simple chemical structure, linear PEI displays intricate solution dynamics, which can be harnessed for environment-sensitive biomaterials and for overcoming current challenges with DNA delivery.

Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets are bound). These difficulties are fundamental, stemming from reliance on base pairing alone to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable molecular conformation change and durable target capture via activatable covalent cross-linking (Vieregg et al, J. Am. Chem. Soc. 2013). In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligonucleotide targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2À3 orders of magnitude. Semi-quantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1À2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications in vitro and in vivo; this talk will present our latest results on SC probe applications.

their bending stiffness is on the order of, and resists, the Brownian forces that randomize their conformation. The study of the collective dynamics of semiflexible assemblies has come to prominence because it underlies the physics of force-transmission and mechanotransduction in cells and tissues. We had previously proposed modeling a semiflexible filament as a string of beams that bend continuously under Brownian forces (Chandran et al, 2009). This idealization not only captures the high-order nonlinear bending of the filament, but it does so at reduced computational cost compared to current string-ofbeads idealizations. We had also proposed solving the relative solvent velocity along the filament as an implicit variable; which is equivalent to including several orders of hydrodynamic interaction and solvent-back reflection in the polymer dynamics (Chandran et al, 2010). In this presentation we compare the predictions of the string-of-beams model with implicit hydrodynamics against that of string-of-beads approaches for new insight on semiflexible polymer dynamics that is produced by the higher-order bending and interaction terms.