Effect of PEG and mPEG-Anthracene on tRNA Aggregation and Particle Formation

Abstract

Poly(ethylene glycol) (PEG) and its derivatives are synthetic polymers with major applications in gene and drug delivery systems. Synthetic polymers are also used to transport miRNA and siRNA in vitro. We studied the interaction of tRNA with several PEGs of different compositions, such as PEG 3350, PEG 6000, and mPEG-anthracene under physiological conditions. FTIR, UV−visible, CD, and fluorescence spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the PEG binding mode, the binding constant, and the effects of polymer complexation on tRNA stability, aggregation, and particle formation. Structural analysis showed that PEG-tRNA interaction occurs via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K PEG 3350-tRNA = 1.9 (±0.5) × 10 4 M −1 , K PEG 6000-tRNA = 8.9 (±1) × 10 4 M −1 , and K mPEG-anthracene = 1.2 (±0.40) × 10 3 M −1 show stronger polymer− RNA complexation by PEG 6000 and by PEG 3350 than the mPEG-anthracene. AFM imaging showed that PEG complexes contain on average one tRNA with PEG 3350, five tRNA with PEG 6000, and ten tRNA molecules with mPEGanthracene. tRNA aggregation and particle formation occurred at high polymer concentrations, whereas it remains in A-family structure. ■ INTRODUCTIONSynthetic polymers play a major role in drug and gene delivery.1−3 Among synthetic polymers, poly(ethylene glycol) and its derivatives show potential applications in gene and drug delivery due to their solubility, nontoxicity, and biocompatibility. 4,5 It has been shown that PEG induces significant changes in DNA solubility and structure under given conditions. 6 DNA concentration, pH, ionic strength of the solution, and the presence of divalent metal ions have been shown to impact PEG-DNA precipitation.6 PEGylation of synthetic polymers such as dendrimers is shown to reduce toxicity and increase biocompatibility and DNA transfection. 4,5,7 Similarly, the effect of PEGylation on the toxicity and permeability of biopolymers such as chitosan has been recently reported. 8,9 Whereas major attention has been focused on polymer−DNA complexes because of their applications in gene delivery, little is known about polymer−RNA interaction. Synthetic polymers can transport miRNA and siRNA in vitro.10−12 The interaction of dendrimers with tRNA has been recently reported. 13 Even though the interactions of PEGylated dendrimers with DNA, RNA, and protein are wellcharacterized, 13−16 detailed structural analysis of PEG and mPEG derivatives complexes with RNA is not known. Therefore, it was of interest to study the interaction of PEGs and mPEGderivatives with tRNA, using spectroscopic methods and AFM imaging.Fluorescence quenching has been used as a technique for quantifying the binding affinities of macromolecules with ligands. 17Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore, induced by a variety of molecular interactions with quencher molecule. Therefore, it is possibl...

Locating the binding sites of antioxidants resveratrol, genistein and curcumin with tRNA

“…2A and B). Similar aggregation and particle formation were observed for tRNA upon complexation with other ligands [27][28][29][30][31].…”

“…1A-C, diff., 1 mM). The polyphenol-base bindings are via guanine and adenine N7 and uracil O2 atoms [27][28][29][30][31].…”

A novel EGFR-targeted gene delivery system based on complexes self-assembled by EGF, DNA, and activated PAMAM dendrimers

“…[2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], were prepared with a fixed final DNA concentration of 50 µg/mL and a total volume of 100 µL. Each mixture was incubated with DNaseI (5 U/µg of plasmid DNA) at 37°C for 1 hour.…”

“…6,[8][9][10][11] Their applications in drug delivery helped optimize in vivo distribution of drugs and passive tumor targeting through the EPR effect. 4 By taking advantage of the modifiable amino groups on the surface, researchers have conjugated different ligands, such as biotinavidin, 12 lysine, 13,14 and PEG [15][16][17] onto PAMAM surfaces to enhance transfection efficiency in vivo and in vitro. Recent studies reveal that PAMAM dendrimers activated by timecontrolled heat treatment can make dendriplexes more flexible and exhibit higher transfection efficiency, 18,19 even though such activation may cause the sacrifice of superficial charges by losing some branches of the dendrimer.…”

Effect of Synthetic Polymers on tRNA Nanoparticle Formation

“…It has been shown that synthetic polymers induce significant changes in DNA and RNA solubility and structure under given conditions [8][9][10][11][12][13][14][15][16][17][18][19][20]. Synthetic polymers are also used to transport miRNA and siRNA in vitro [11][12][13][14][15][16][17][18][19][20][21][22][23][24]. PEGylation of synthetic polymers such as dendrimers is shown to reduce toxicity and increase biocompatibility and DNA Transfection [6,7,9].…”