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

Abstract

An amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-N-acryloxysuccinimide)) (poly(TBAm-b-(NAM/NAS)) and a random copolymer poly(NAM/NAS), synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization process, have been used as support for oligonucleotide (ODN) synthesis, to elaborate polymer-oligonucleotide conjugates. In a first step, starters of ODN solid-phase synthesis were coupled to activated ester functions of polymers, and second, resulting functionalized polymers were covalently grafted onto hydroxylated controlled pore glass (CPG) support to further accomplish ODN synthesis. An efficient capping of residual hydroxyl functions of CPG was performed before synthesis, with both acetic anhydride and diethoxy-N,N-diisopropyl-phosphoramidite reagents, to suppress parasite-free ODN population present in conjugate crude material and resulting from syntheses directly initiated on silica beads. After purification, conjugates were evaluated in a DNA hybridization assay on a microarray, as macromolecules being able to favor capture of the target. Conjugate coating conditions were studied on the dT25/dA25 model. The role of the hydrophobic part (poly(TBAm)) of the conjugate synthesized with the block copolymer in the orientation of the conjugate after coating was revealed by spotting experiments achieved in a mixed solvent (DMF/H(2)O). The use of block copolymer-dT25 conjugate afforded a significant sensitivity improvement of the hybridization assay.

Living Radical Polymerization by the RAFT Process—A First Update

“…[244] Copolymers of N-(meth)acryloyl succinimide or pentafluorophenyl methacrylate (these monomers, NAS, NMS, and PFMA are referred to as active esters of (meth)acrylates) have been produced by RAFT polymerization and served as substrates for biofunctionalization or other grafting reactions using grafting-to processes. [66,73,245,246] Recent examples include poly(t-butyl acrylamide-block-poly(Nacryloylmorpholine-co-NAS) [245] poly(DMA-co-NAS), [246] PFMA-block-PNAM, [73] and PFMA-block-PMMA. [73] …”

Effect of Chemical Crosslinking on Properties of Polymer Microbeads: A Review

“…Biological cross-linking is also an emerging cross-linking method wherein biomolecules such as complementary oligonucleotides [36], oppositely charged peptide [37], and heparin growth factor are used to obtain biological cross-linking [38]. The oligonucleotides such as oligo(cholesteryl) methacrylaten-PEG-oligo(cholesteryl)methacrylate)n-(OC 5 ma-PEO-C 5 MA) and oligo(10-cholesteryloxy decyl methacrylate)n-PEG-oligo(10-cholesteryloxy decyl methacrylate)n-OC 10 MA-PEO-OC 10 MA) were used [36].…”

“…The oligonucleotides such as oligo(cholesteryl) methacrylaten-PEG-oligo(cholesteryl)methacrylate)n-(OC 5 ma-PEO-C 5 MA) and oligo(10-cholesteryloxy decyl methacrylate)n-PEG-oligo(10-cholesteryloxy decyl methacrylate)n-OC 10 MA-PEO-OC 10 MA) were used [36]. Further, peptide containing opposite charge can be used to obtain biological cross-linked polymers [38].…”

Experimental Requirements for an Efficient Control of Free‐Radical Polymerizations via the Reversible Addition‐Fragmentation Chain Transfer (RAFT) Process

“…When the nature of the two blocks is similar, the choice of the sequence is less crucial. Each block can be synthesized [9,67,119,356] BMA b) [151] EHMA b) [151] Sty [119] b) [151,229] MA [356] VAc [67] -C(Me) 2 CO 2 R 0d) Sty [356]c) [234] NIPAm c) [281] DMAm [343] -C(Me) 2 CO 2 CH 2 CH 2 C 6 F 13 MMA [113] -C(Me) 2 CONHC 18 H 37 NIPAm [347] -C(Me) 2 CONHCH 2 CH 2 OH MMA [119] -C(Me) 2 CONHCH 2 CH 2 SO 3 H MAETMAC [160] VPPS [160] MAm [160] DMAPMAm [160] MSASP [160] -C(Me)(SPh)CO 2 Et Sty [19,316] MA [19] -C(Me)(CO 2 Et) 2 AA [248] NAM [43] -C(Me) 3 MMA [9,119] Sty b) [230] AA [248] VC 2 a) [256] StyOCOMe [240] MA a) [256] BA [387] tBA [262] NAS a) [265][266][267][268][269][270][271] DMAm …”