ac1033769_si_001.pdf (1.19 MB)
Terminal Protection of Small Molecule-Linked DNA: A Versatile Biosensor Platform for Protein Binding and Gene Typing Assay
journal contributionposted on 2011-04-15, 00:00 authored by Zhan Wu, Hongqi Wang, Min Guo, Li-Juan Tang, Ru-Qin Yu, Jian-Hui Jiang
Assays of small molecule−protein interactions are of tremendous importance in chemical genetics, molecular diagnostics, and drug development. This work reports a new finding of generalized terminal protection that small molecule-DNA chimeras are protected from degradation by various DNA exonucleases, when the small molecule moieties are bound to their protein targets. This generalization converts small molecule−protein interaction assays into the detection of DNA of various structures, affording a useful mechanism for the analytics of small molecules. On the basis of this mechanism, a label-free biosensor strategy has been developed for a homogeneous assay of protein−small molecule interactions based on the fluorescence staining detection. Also, a label-free SNP genotyping technique is proposed based on polymerase extension of a single nucleotide with a small molecule label. The developed techniques are demonstrated using a model protein−small molecule system of biotin/streptavidin and a model SNP system of human β-globin gene around the position of codon 39. The results revealed that the protein−small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 100 nM with a detection limit of 0.1 nM, and the SNP typing technique gives dynamic responses in the concentration range from 0.1 to 200 nM with a detection limit of 0.02 nM. Besides desirable sensitivity, the developed strategies also offer high selectivity, excellent reproducibility, low cost, and simplified operations, implying that these techniques may hold considerable potential for molecular diagnostics and genomic research.