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

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 N-acryloylmorpholine (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 x 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. (C) 2004 Wiley Periodicals, Inc.