Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

Abstract

ABSTRACT:A water-soluble statistical poly(N-acryloylmorpholine-co-N-acryloxysuccinimide) [poly(NAM/NAS)] copolymer was studied for polymer-oligonucleotide (ODN) conjugate elaboration and for further use in diagnostic applications. Three different copolymers were first prepared by free-radical solution polymerization with different Nacryloylmorpholine (NAM) and N-acryloxysuccinimide (NAS) molar ratios (80/20, 70/30, and 60/40). Their number-average molecular weights ranged from 98,000 to 120,000 g/mol, as determined by aqueous size exclusion chromatography with an online light-scattering detector. Then, polymer-ODN conjugates were obtained via a strategy consisting of the direct synthesis of ODNs onto polymer chains previously grafted onto a controlled pore glass support. Before the grafting of the polymer onto the solid support, a preliminary step was performed to bind a nucleotide starter along the polymer chain (via the reactive NAS units) to initiate automated DNA synthesis. To multiply the number of ODNs growing from starters, a branched phosphoramidite synthon [bearing two O-dimethoxytrityl groups] was introduced at the first step of ODN elongation as a short sequence of four branched synthons alternated with three thymidine residues. Conjugates were assessed in a DNA sandwich hybridization test developed for hepatitis B virus detection. Sensitivity limits were evaluated and compared to those obtained with an other polymer, poly(maleic anhydride-alt-methyl vinyl ether) [poly(MA/MVE)]. A sensitivity limit of 2.6 ϫ 10 7 DNA copies/mL was reached with the poly(MA/MVE)-ODN conjugate at the capture phase and with the poly(NAM/NAS)-branched ODN conjugate at the detection phase of the test.

Characterization and electrical properties of methyl‐2‐hydroxyethyl cellulose doped with erbium nitrate salt

“…McCormick and coworkers114 used NHS‐A in the presence of a macro‐RAFT agent bearing a PEO block to obtain poly(ethylene oxide)‐ b ‐poly(DMA‐ co ‐NHS‐A) diblock polymers or poly(ethylene oxide)‐ b ‐poly(DMA‐ co ‐NHS‐A)‐ b ‐poly(NIPAAm) triblock polymers. The versatility of these activated ester functional polymer precursors was demonstrated for several applications, such as the attachment of peptide, DNA, or fluorescent dye 115–117. The NHS‐A units were also modified in the presence of ethylene diamine or N,N ‐dimethyl ethylene diamine to give RAFT‐functional polymers bearing primary or secondary amine pendant groups.…”

Polymer−Oligonucleotide Conjugate Synthesis from an Amphiphilic Block Copolymer. Applications to DNA Detection on Microarray

“…As previously described (14), polymer grafting onto functionalized CPG was optimum after 5 days reaction. Grafting efficiency of the polymer-starter 1 conjugate was controlled through quantification of dimethoxytrityl groups released from starters 1 bound to polymer chain.…”

“…Starter 1 Synthesis. Starter 1 structure was designed in order to avoid different hurdles encountered in this laboratory with other molecules elaborated for the same purpose: (i) a nucleotide analogue (5′-dimethoxytrityl-2′-deoxythymidine-3′-(6-aminohexyl phosphate)) which was described to react with maleic anhydride copolymers (13) or poly(NAM/NAS) copolymers (14) not only via the terminal amino group but also via the intracyclic amine function of the nucleic base, forming an unstable link (in ammonia) between polymer and starter, (ii) a neutral starter, DMT-O(CH 2 ) 6 NH 2 , efficiently coupled to poly-(NAM/NAS) but appearing to inhibit further grafting reaction onto CPG (step B) (unpublished results). According to this latter observation, we aimed at introducing one phosphate group (bringing a charge) in the alkyl chain of the molecule, to restore the grafting ability of the resulting starter 1-polymer conjugate onto hydroxylated silica support.…”

Building nanostructures using RAFT polymerization

“…The versatility of these activated ester functional polymer precursors was demonstrated for several applications, such as the attachment of peptide, DNA, or fluorescent dye. [115][116][117] The NHS-A units were also modified in the presence of ethylene diamine or N,N-dimethyl ethylene diamine to give RAFT-functional polymers bearing primary or secondary amine pendant groups. The methacrylate analogue to NHS-A, N-methacryloxysuccinimide (NHS-MA), has also been polymerized using RAFT, however, polymerization control proved difficult (a broad polydispersity was observed).…”