DNA-mounted self-assembly: new approaches for genomic analysis and SNP detection.

This article presents an overview of new emerging approaches for nucleic acid detection via hybridization techniques that can potentially be applied to genomic analysis and SNP identification in clinical diagnostics. Despite the availability of a diverse variety of SNP genotyping technologies on the diagnostic market, none has truly succeeded in dominating its competitors thus far. Having been designed for specific diagnostic purposes or clinical applications, each of the existing bio-assay systems (briefly outlined here) is usually limited to a relatively narrow aspect or format of nucleic acid detection, and thus cannot entirely satisfy all the varieties of commercial requirements and clinical demands. This drives the diagnostic sector to pursue novel, cost-effective approaches to ensure rapid and reliable identification of pathogenic or hereditary human diseases. Hence, the purpose of this review is to highlight some new strategic directions in DNA detection technologies in order to inspire development of novel molecular diagnostic tools and bio-assay systems with superior reliability, reproducibility, robustness, accuracy and sensitivity at lower assay cost. One approach to improving the sensitivity of an assay to confidently discriminate between single point mutations is based on the use of target assembled, split-probe systems, which constitutes the main focus of this review.

Biophysical and X-ray crystallographic analysis of Mps1 kinase inhibitor complexes.

The dual-specificity protein kinase monopolar spindle 1 (Mps1) is a central component of the mitotic spindle assembly checkpoint (SAC), a sensing mechanism that prevents anaphase until all chromosomes are bioriented on the metaphase plate. Partial depletion of Mps1 protein levels sensitizes transformed, but not untransformed, human cells to therapeutic doses of the anticancer agent Taxol, making it an attractive novel therapeutic cancer target. We have previously determined the X-ray structure of the catalytic domain of human Mps1 in complex with the anthrapyrazolone kinase inhibitor SP600125. In order to validate distinct inhibitors that target this enzyme and improve our understanding of nucleotide binding site architecture, we now report a biophysical and structural evaluation of the Mps1 catalytic domain in the presence of ATP and the aspecific model kinase inhibitor staurosporine. Collective in silico, enzymatic, and fluorescent screens also identified several new lead quinazoline Mps1 inhibitors, including a low-affinity compound termed Compound 4 (Cpd 4), whose interaction with the Mps1 kinase domain was further characterized by X-ray crystallography. A novel biophysical analysis demonstrated that the intrinsic fluorescence of SP600125 changed markedly upon Mps1 binding, allowing spectrophotometric displacement analysis and determination of dissociation constants for ATP-competitive Mps1 inhibitors. By illuminating the structure of the Mps1 ATP-binding site our results provide novel biophysical insights into Mps1-ligand interactions that will be useful for the development of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.