Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly (N-isopropylacrylamide)

A versatile strategy has been developed for selectively and sequentially is olating targets in a liquid-phase affinity separation environment. The stra tegy uses a recently developed approach for joining together molecules in l inkages that are defined by the complementary pairing of oligonucleotides c onjugated to the different molecules [Niemeyer, C. M., Sane, T., Smith, C. L., and Canter, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work pr esented here, streptavidin was noncovalently coupled with the temperature-r esponsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-s pecific hybridization of oligonucleotides conjugated to the protein and pol ymer. A 20-mer oligonucleotide was covalently linked through a heterobifunc tional linker to a genetically engineered streptavidin variant that contain ed a unique cysteine residue at the solvent-accessible site Glu 116. The co mplementary DNA sequence was conjugated to the end of a linear eater-activa ted poly(NIPAAM). The two conjugates were allowed to self-assemble in solut ion via hybridization of their complementary DNA sequences. The streptavidi n-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled b iotin or biotinylated alkaline phosphatase above 32 degrees C through the t hermally induced phase separation activity of the poly(NIPAAM). The strepta vidin-oligo species could then be reversibly separated from the precipitate d 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 se lective precipitation of different polymers and streptavidin complexes base d on the sequence-specific hybridization of their oligonucleotide appendage s.