DNA Hybridization in Thermoresponsive Polymer Nanoparticles

Abstract

We achieve very high hybridization efficiencies by using a new method to immobilize DNA strands on the surface of thermoresponsive polymer nanoparticles. Hybridization efficiencies of about 70 % are obtained between the DNA immobilized in the particles and a complementary strand in solution, even at very low ionic strengths (1 mM). The polymer nanoparticles have a glassy poly(methylmethacrylate) (PMMA) core and a thermoresponsive shell of poly(N-isopropylacrylamide) (PNIPAM) containing positive charges. After a DNA strand labeled with a fluorescence probe is loaded onto the particles at room temperature, the temperature is increased above the volume phase transition temperature of the PNIPAM shell, TVPT approximately 28 degrees C. The collapse of the particle shell immobilizes the DNA while maintaining its availability for hybridization with a complementary strand. Förster resonance energy transfer (FRET) is used to detect the hybridization with a complementary DNA strand labeled with a FRET acceptor probe.

Polymeric Nanoparticle Engineering: From Temperature-Responsive Polymer Mesoglobules to Gene Delivery Systems

“…52 Importantly, analogous to peptide−PNIPAM systems, DNA and PNIPAM are compatible only in chilled aqueous medium. This was confirmed by recent studies of Moura et al 53 and Martinho et al 54 who were able to immobilize oligonucleotide (OND) and DNA on thermoresponsive shell of PNIPAM, copolymerized with a small amount of positively charged comonomer, aminoethyl methacrylate hydrochloride, in order to increase the absorption of negatively charged DNA and OND. At temperatures lower than PTT, the nucleotides are distributed into the interior of the shell.…”

“…By increasing the temperature above the phase transition, DNA and OND are anchored in the dense surface layer, not located inside the shell but oriented toward the water phase. Other results, published elsewhere, 53,54 suggest that the incorporation into the TRP of small amounts of hydrophobic, weakly acidic, or basic comonomers is an important factor for successful biomedical and biotechnological applications. In that case, the electrostatic interactions were the dominant force.…”

Stimuli‐Responsive magnetic nanoparticles for monoclonal antibody purification

“…Stimuli‐responsive colloidal particles have described applications in areas as diverse as the stabilization, destabilization and inversion of colloidal emulsions, foams and suspensions; catalysis; sensors and drug‐delivery [2, 3]. Curiously, few accounts describe the application of stimuli‐responsive nanoparticles in biomolecules separation [4–12], with no reports published, to our knowledge, on mAb purification from mammalian cell cultures. However, thermo‐responsive magnetic particles immobilized with bovine serum albumin (BSA) have been used for the affinity purification of anti‐BSA antibodies from rabbit antiserum [13], while systems envisioned for immunoassays have also used thermo‐responsive particles in antibody‐antigen recognition [14, 15].…”

A Cationic Smart Copolymer for DNA Binding

“…Since the technique measure the diffusion coefficients of the fluorescent species only, there is no interference of the non-fluorescent free copolymer. Previously characterized dT 25 oligonucleotides labeled at the 5′-terminus with a rhodamine X [27,28,29] showed that the probe has a high quantum yield and good photostability. FCS measurements of the 1:1 and 2:1 SRP:DNA-ROX mixtures yield the diffusion coefficients of the samples, from which the hydrodynamic radius ( R FCS ) can be calculated using the Stokes-Einstein equation.…”