S Patrick Walton | PubMed Health

PubMed2006

Analysis of hybridization on the molecular barcode GeneChip microarray.

Microarrays have been developed for analysis of transcriptional profiles in many organisms. For experimental simplicity and systems for which microarrays do not exist, it would be desirable to use a standard microarray platform for the analysis of multiple systems. The molecular barcode (MB) Affymetrix GeneChip could serve as such a platform. The reproducibility and quantitative capacity of the MB GeneChip were examined. Using mixed PCR templates of defined template quantity, the individual concentration responses of 384 array features were measured and shown to be highly reproducible. Moreover, the binding behaviors of the mismatched array features mirror those of the matched features, at reduced intensity. Additional analysis defined the importance of particular sequence motifs in the prediction of high-affinity and low-affinity target hybridization. The data support the future application of MB GeneChips for quantitative applications. It is proposed that at least seven orders-of-magnitude in accurate concentration sensitivity could be achieved.

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PubMed2002

Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA.

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit beta-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.

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