Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).
A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.
Fong
R B
RB
Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
Ding
Z
Z
Long
C J
CJ
Hoffman
A S
AS
Stayton
P S
PS
D000178:Acrylamides / Q000138:chemical synthesis* / Q000737:chemistry; D000180:Acrylic Resins; D000469:Alkaline Phosphatase / Q000737:chemistry; D000838:Anions; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002627:Chemistry, Physical; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry; D006358:Hot Temperature; D007202:Indicators and Reagents; D009841:Oligonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D012996:Solutions; D019809:Streptavidin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification
D000178:Acrylamides; D000180:Acrylic Resins; D000838:Anions; D007202:Indicators and Reagents; D009841:Oligonucleotides; D012996:Solutions; C119790:streptavidin-poly(N-isopropylacrylamide); D001710:Biotin; D004247:DNA; D019809:Streptavidin; D000469:Alkaline Phosphatase