Affinity capture-facilitated preparation of aequorin- oligonucleotide conjugates for rapid hybridization assays.

We report a general procedure for the preparation of biomolecular conjugates that combine the molecular recognition properties of oligonucleotides with the high detectability of the photoprotein aequorin. Central to the conjugation protocols is the use of recombinant aequorin fused to a hexahistidine tag. In one protocol, an amino-modified oligonucleotide was treated with a homobifunctional cross-linker carrying two N-hydroxysuccinimide ester groups, and the derivative was allowed to react with (His)(6)-aequorin. A second strategy involved the introduction of protected sulfhydryl groups into (His)(6)-aequorin and subsequent reaction with a heterobifunctional linker containing a N-hydroxysuccinimide and a maleimide group. The strong, but reversible, binding of (His)(6)-aequorin to Ni(2+)-nitrilotriacetic acid agarose enabled the rapid and effective removal of the unreacted oligonucleotide, which otherwise diminishes the performance of the hybridization assay by competing with the conjugate for the complementary target sequence. Aequorin-oligo conjugates prepared by affinity capture showed similar performance with those purified by anion-exchange HPLC. The conjugates were applied to the development of rapid bioluminometric hybridization assays. The analytical range extended from 2 to 2000 pmol/L of target DNA. The reproducibility was less than 10%. The conjugate obtained from a reaction of 10 nmol of (His)(6)-aequorin is sufficient for about 5000 hybridization assays. The proposed conjugation strategy is general because a variety of reporter proteins can be fused to hexahistidine tag by using suitable vectors that are commercially available.