Polymer-oligonucleotide conjugate synthesis from an amphiphilic block copolymer. Applications to DNA detection on microarray.
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.
de Lambert
Bertrand
B
Unité Mixte CNRS/bioMérieux, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon Cedex 07, France.
Chaix
Carole
C
Charreyrex
Marie-Thérèse
MT
Laurent
Alain
A
Aigoui
Arnaud
A
Perrin-Rubens
Agnès
A
Pichot
Christian
C
D000577:Amides; D002021:Buffers; D003432:Cross-Linking Reagents; D057927:Hydrophobic and Hydrophilic Interactions; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D010756:Phosphoric Acids; D011108:Polymers / Q000737:chemistry*
D000577:Amides; D002021:Buffers; D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D010756:Phosphoric Acids; D011108:Polymers; C011067:phosphoramidic acid