Complexes of DNA with cationic peptides: Conditions of formation and factors effecting internalization by mammalian cells

Abstract

This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene.

TAT-conjugated nanoparticles for the CNS delivery of anti-HIV drugs

“…The above phenomenon has been observed by others with TAT-conjugated macromolecular therapeutics. For example, Dizhe et al have reported increased cellular uptake of TAT/DNA complex in the presence of excess free TAT-peptide, and has attributed to a stimulatory effect of excess TAT-peptide on the cellular entry of TAT/DNA conjugate [42]. …”

Insulin-Mediated Downregulation of Apolipoprotein A-I Gene in Human Hepatoma Cell Line HepG2: The Role of Interaction Between FOXO1 and LXRβ Transcription Factors

“…The pCMVLacZ [Dizhe et al, 2006] and pA1(À256/þ72) [Lapikov et al, 2008] plasmids were described elsewhere. pCMVHNF4a, the expression vector of human transcription factor HNF4a, was a generous gift of Dr. Fukamizu (University of Tsukuba, Tsukuba, Japan).…”

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery

“…The plasmid DNA was extracted by phenol:chloroform: isoamyl alcohol (25:24:1) pH 8 and precipitated with ethanol. The dried DNA pellet was reconstituted with TAE buffer and subjected to agarose gel electrophoresis [9,46].…”

“…Heparin has a greater charge density compared to heparan sulfate [47] and is a stronger polyanion than DNA. Heparin is therefore, capable of displacing DNA from its complexes with polycations [46]. The peptide-DNA complexes responded differently on encountering heparin.…”