Intrinsic Dynamics of DNA–Polymer Complexes: A Mechanism for DNA Release

Abstract

The transfer of genetic material into cells using non-viral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, linewidth, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR timescale (< millisecond). The dendrimers retain a high degree of mobility in the polyplex, whereas the DNA shows restrained mobility, suggesting that the polyplex is a highly dynamic complex with a rapidly exchanging dendrimer atmosphere around a more rigid DNA framework.

Quantitative Measurement of Cationic Polymer Vector and Polymer–pDNA Polyplex Intercalation into the Cell Plasma Membrane

“…Our group previously investigated the G5 PAMAM-DNA interaction using NMR and showed it to be highly dynamic, consisting of rigid DNA and mobile dendrimer. 28 This may be the reason why the G5 PAMAM polyplexes have a similar zeta potential as the free G5 PAMAM. Similar NMR studies using L-PEI polyplexes may help determine if the DNA-polymer interactions are different in those polyplexes.…”

Rapid Exchange Between Free and Bound States in RNA–Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release

“…For a X-H group, R 2 relaxation rates are dependent on a linear combination of spectral density functions evaluated at five frequencies, J(0), J(ω H ), J(ω X ), J(ω X + ω H ), and J(ω X − ω H ), where ω H and ω X are the frequencies of H and X nuclei respectively at a given spectrometer field. 54 Assuming that dipole-dipole coupling dominates 1 H R 2 relaxation due to directly bonded carbon as well as neighboring protons within 4 Å, R 2,polyplex was expressed as a sum of R 2 relaxation rates 31,39,55 $${R_{2,\mathit{polyplex}}}^{\mathrm{H}}={{\displaystyle \sum }}^{}\frac{1}{8}{d}^2\left[4\mathrm{normalJ}\left(0\right)+3\mathrm{normalJ}\left({\mathrm{normal\omega }}_H\right)+\mathrm{normalJ}\left({\mathrm{\omega }}_X-{\mathrm{\omega }}_H\right)+6\mathrm{normalJ}\left({\mathrm{normal\omega }}_X\right)+6\mathrm{normalJ}\left({\mathrm{\omega }}_X+{\mathrm{\omega }}_H\right)\right]$$where d is the dipolar coupling constant given by $$d=\frac{{\mathrm{\mu }}_0{\gamma }_H{\gamma }_{X}h}{8{\pi }^2\langle {r^3}_{\mathit{HX}}\rangle }$$μ 0 is the permittivity of free space, γ H and γ C are the magnetogyric ratios of 1 H and 13 C respectively and h is the Plank’s constant. r HC is the distance of the aromatic and sugar protons to their directly bonded carbon (1.104 Å and 1.115 Å for base C–H bond and sugar C1′–H1′ bond respectively) and r HH are the distances to neighboring protons within 4 Å.…”

“…31 The G5 PAMAM scaffold was PEGylated to increase polyplex solubility and this form has been shown to be effective for in vivo knockdown of protein expression. 32 Based on the 1 H NMR chemical shifts, there was no evidence for changes in the DNA duplex structure or dynamics upon binding to the dendrimer.…”

“…31 TAR is one of the key regulatory RNA elements in the HIV-1 viral genome which plays essential roles in viral replication. Although there has been interest in studying TAR/PAMAM dendrimer interactions in the context of Tat-TAR inhibition, 36,37 here we focus on using TAR as a model flexible RNA system 38–41 that contains non-canonical secondary structural elements, including a 3-nucleotide bulge and 6-nucleotide apical loop.…”

Complex coacervates of hyaluronic acid and lysozyme: Effect on protein structure and physical stability

“…A wide variety of complex coacervates systems have been reported and investigated in the literature, e.g. protein-protein [5,32], protein-polymer [13,31], nucleic acid-polymer [16,26,24] and nucleic acid-dendrimer [11,19]. A more complete overview can be found in recent review papers [14,30].…”