Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly (N-isopropylacrylamide)
Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly (N-isopropylacrylamide)
复制标题
DOI：
10.1021/bc980151f
复制
发表时间：
1999-09-01
影响因子：
4.7
通讯作者：
Stayton, PS
中科院分区：
文献类型：
--
作者：
Fong, RB;Ding, ZL;Stayton, PS
已经开发了一种通用的策略，用于在液相亲和分离环境中选择性地和顺序地分离靶标。该策略使用最近开发的方法将分子以连接方式连接在一起，所述连接由缀合至不同分子的寡核苷酸的互补配对限定[Niemeyer，C. M.，Sane，T.，史密斯角，澳-地L.，和Canter，C. R.（1994）Nucleic Acids Res.22，5530-9]。在这里提出的工作中，链霉亲和素与温度响应性聚（N-异丙基丙烯酰胺）[聚（NIPAAM）]通过偶联到蛋白质和聚合物的寡核苷酸的序列特异性杂交非共价偶联。一个20聚体的寡核苷酸共价连接，通过异双功能接头的基因工程链霉亲和素的变体，含有一个独特的半胱氨酸残基在溶剂可及位点Glu 116。将互补DNA序列缀合至线性酯活化的聚（NIPAAM）的末端。允许两种缀合物通过其互补DNA序列的杂交在溶液中自组装。链霉亲和素-聚（NIPAAM）复合物可用于在32 ℃以上通过聚（NIPAAM）的热诱导相分离活性亲和沉淀放射性标记的生物素或生物素化碱性磷酸酶。链霉亲和素-寡核苷酸物质然后可以从沉淀的聚合物-寡核苷酸缀合物中可逆地分离，并通过降低盐浓度而再循环，这导致短双链DNA连接的变性。使用寡核苷酸将聚合物偶联至链霉亲和素允许基于它们的寡核苷酸附件的序列特异性杂交选择性沉淀不同的聚合物和链霉亲和素复合物。
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., Sane, T., Smith, C. L., and Canter, 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 eater-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.