Thermoprecipitation of Streptavidin via Oligonucleotide-Mediated Self-Assembly with Poly(N-isopropylacrylamide)

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Abstract
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 °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.