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Sequence‐specific affinity precipitation of oligonucleotide using poly( N ‐isopropylacrylamide)–oligonucleotide conjugate
Author(s) -
Mori Takeshi,
Umeno Daisuke,
Maeda Mizuo
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20010205)72:3<261::aid-bit2>3.0.co;2-7
Subject(s) - conjugate , oligonucleotide , chemistry , poly(n isopropylacrylamide) , precipitation , aqueous solution , residue (chemistry) , chromatography , polymer , combinatorial chemistry , polymer chemistry , copolymer , stereochemistry , dna , organic chemistry , biochemistry , mathematics , mathematical analysis , physics , meteorology
In this study we develop a sequence‐specific precipitation separation system of oligonucleotide (ODN) using a conjugate between poly( N ‐isopropylacrylamide) (PNIPAM) and ODN. PNIPAM is known as a thermoresponsive polymer and dehydrates to precipitate above its phase transition temperature in an aqueous milieu. The principal advantage of this separation system using the conjugate is that the hybridization reaction between the conjugate and oligonucleotide is conducted in homogeneous solution. The conjugate was prepared by copolymerization between N ‐isopropylacrylamide and a vinyl‐derivatized (dT) 8 . The obtained conjugate efficiently precipitated (dA) 8 from solution when the solution contained more than 1.5 M NaCl. The conjugate containing 3 nmol of (dT) 8 residue was able to precipitate 1.4 nmol of (dA) 8 , suggesting that the (dT) 8 residue of the conjugate formed a triple helix with (dA) 8 . From an equimolar mixture of (dA) 8 and its one point mutant, the conjugate selectively precipitated (dA) 8 : the highest selectivity was obtained for the isolation of (dA) 8 from the mixture consisting of (dA) 4 dT(dA) 3 and (dA) 8 . When the conjugate was applied for the precipitation of five oligo(dA)s having different chain lengths, the longer oligo(dA)s tended to be precipitated by the conjugate more efficiently than the shorter ones. The conjugate could be used repeatedly for precipitation of (dA) 8 without showing any loss in precipitation efficiency. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 261–268, 2001.